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University of Szeged Pharmaceutical Analysis Practicals Edited by: György Dombi Gerda Szakonyi Authors: György Dombi Éva Kalmár Gerda Szakonyi Henriett Diána Szűcs Reviewed by: Krisztina Novák-Takács Szeged, 2015 This work is supported by the European Union, co-financed by the European Social Fund, within the framework of "Coordinated, practice-oriented, student-friendly modernization of biomedical education in three Hungarian universities (Pécs, Debrecen, Szeged), with focus on the strengthening of international competitiveness" TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project. The curriculum can not be sold in any form! TABLE OF CONTENTS CONDUCTOMETRY ....................................................................................................................... 3 CONDUCTOMETRIC TITRATION OF CARBOXYLIC ACIDS .............................................................. 7 ANALYSIS OF ACETYLSALICYLIC ACID ....................................................................................... 8 ANALYSIS OF BENZOIC ACID ........................................................................................................ 9 POTENTIOMETRIC (pH-METRIC) TITRATIONS ............................................................................ 10 HOW TO USE THE GLASS ELECTRODE ....................................................................................... 15 POTENTIOMETRIC TITRATION .................................................................................................... 15 EVALUATION OF THE MEASUREMENT ....................................................................................... 15 QUANTITATIVE ASSAYS BY TITRATION WITH ALKALINE SOLUTIONS........................................ 16 DINATRII PHOSPHAS DIHYDRICUS ............................................................................................. 17 NATRII DIHYDROGENOPHOSPHAS DIHYDRICUS ......................................................................... 18 CHININI HYDROCHLORIDUM ..................................................................................................... 19 UNGUENTUM AD VULNERA ....................................................................................................... 21 SPECTROPHOTOMETRY .............................................................................................................. 23 PULVIS CHINACISALIS CUM VITAMINO C .................................................................................. 33 TABLETTA ASPIRINI 500 (ASPIRIN TABLET 500) ....................................................................... 37 SUPPOSITORIUM PARACETAMOLI 500 MG.................................................................................. 39 SPARSORIUM ANTISUDORICUM ................................................................................................. 41 SOLUTIO METRONIDAZOLI ........................................................................................................ 43 PULVIS CHOLAGOGUS ............................................................................................................... 44 DETERMINATION OF PROTEIN CONCENTRATION WITH THE BIURET REAGENT .......................... 47 ATOMIC ABSORPTION SPECTROMETRY ..................................................................................... 49 DETERMINATION OF MAGNESIUM CONTENT OF SPARSORIUM ANTISUDORICUM BY FLAME ATOMIC ABSORPTION................................................................................................................ 53 DETERMINTION OF MAGNESIUM CONTENT OF PULVIS NEUTRACIDUS BY FLAME ATOMIC ABSORPTION ............................................................................................................................. 54 DETERMINATION OF ACTIVE INGREDIENTS OF PANADOL EXTRA BY HPLC .............................. 55 COMPLEXOMETRIC TITRATIONS ................................................................................................ 58 PULVIS NEUTRACIDUS ............................................................................................................... 63 SUSPENSIO ZINCI AQUOSA ........................................................................................................ 65 ARGENTOMETRIC ANALYSIS ..................................................................................................... 66 SPARSORIUM SULFABORICUM ................................................................................................... 67 REDOX TITRATIONS .................................................................................................................. 68 SUPPOSITORIUM ANTIPYRETICUM PRO INFATE VEL PRO PARVULO ........................................... 74 INJECTIO ALGOPYRINI 50% ....................................................................................................... 76 ACIDBASE TITRATIONS ........................................................................................................... 77 SPIRITUS IODOSALICYLATUS ..................................................................................................... 80 TEST YOURSELF – SAMPLE TEST QUESTIONS ............................................................................ 82 APPENDIX .............................................................................................................................. 92 UNICAM UV/VIS SPECTROPHOTOMETER MANUAL .................................................................. 93 UV-1601 SHIMADZU SPECTROPHOTOMETER MANUAL ............................................................. 95 MARS CEM MICROWAVE DESTRUCTOR MANUAL ..................................................................... 97 ATOMIC ABSORPTION SPECTROMETER MANUAL ...................................................................... 98 HPLC MANUAL ...................................................................................................................... 100 NMR SPECTRA........................................................................................................................ 109 2 CONDUCTOMETRY (MEASUREMENT OF SPECIFIC CONDUCTANCE) Conductometry is based on the measurement of the conductance of electrolyte solutions. The passage of electric current through a chemical cell is carried out by the ionic species in the solution. It is an additive property, with the participation of all of the ions in the solution. The conductance is specified by the measurement of the resistance of a certain segment of the solution. The conductance (G) is the reciprocal of the resistance (R), its unit is 1/Ω Siemens; S): G= 1 R The conductance is directly proprotional to the surface area (A) of the electrodes and inversely proportional to the distance (d) between the electrodes: 1 A =κ R d κ is the specific conductance, where the resistance of the solution is measured between two electrodes of 1 cm2 area 1 cm apart. The conductance depends on the number of ions in the solution and on the identity of the ions. Some ions move faster than others in an electric field, and their mobility is therefore an important factor too. Dilution of an electrolyte solution will decrease the specific conductance: the lower number of ions present in a given volume, the lower the current flow is. The molar specific conductance () was introduced to characterize of the conductance of certain ions: = 1000 c where c is the concentration of the electrolyte solution. The ions in an infinitely dilute solution contribute to the conductance independently from each other, and the molar specific conductance of an infinite dilute solution can therefore be calculated by summing the conductances of each of the ions in the solution: and are the conductances of cations and anions, respectively in infinitely dilute solution. 3 The electrode Special eletrodes are used during conductometric measurements. The conductance is determined by measurement of the resistance of the solution in a certain volume between two electrodes made of platinized platinum. The surface area of the electrodes is increased and the polarization resistance is decreased by platinization. The electrodes are fixed tightly in a cylindrical unit. The fixed geometry specifies the distance of the electrodes during both the calibration and the measurement. Alternate current is used for the conductometric analysis so as to avoid disturbing electrode processes. A Wheatstone bridge is used to measure the resistance. Concentration measurement (direct conductometry) and conductometric titrations (indirect conductometry) are distinguished in conductometry. In direct conductometry, the concentration is determined by the measurement of conductance. This method is used, for example, to check Aqua purificata or Aqua destillata. An electrode is built into the ion-exchange system that continuously monitors the conductance of ion-exchanged water. When the conductance is above a given limit, the system must be regenerated. According to the European Pharmacopoeia 8th Edition, the maximum allowed conductance of Aqua purificata is 4.3 μS/cm, while that of Aqua ad iniectabilia is 1.1 μS/cm. Conductometric titrations can be applied when the ion concentration changes during a reaction, or when the ion concentration remains constant, but the mobility of the ions changes. Types of conductometric titrations Acid–base titrations It is easy to determine the equivalence point in these titrations because hydrogen ions (H ) are the most mobile of all ions, and hydroxide ions (OH-) are the second most mobile, and the mobilities are well above those of other ions. + 1. Titration of a strong acid with a strong base The titration of hydrochloric acid (HCl) with sodium hydroxide (NaOH) may serve as an example. 4 The neutralization of HCl does not change the electrolyte concentration of the solution before the equivalence point because the H+ are replaced by sodium ions (Na+). The lower mobility of the Na+ results in decreased conductivity. There are two reasons why the conductivity increases after the equivalence point. The excess NaOH increases the electrolyte concentration in the solution, and the mobility of the OH- is high. HCl + NaOH→H2O + NaCl 2. Titration of a weak acid with a strong base As an example, acetic acid may be titrated with NaOH. At the beginning of the titration, the dissociation of acetic acid is blocked by the acetate ions. The H+ concentration is decreased, and the conductance is therefore also decreased, so that a minimum is visible in the titration plot. The concentration of acetate ions increases on the addition of NaOH, and the conductance increases too then slowly. Na+ also contribute to the increase of conductance. The conductance increases sharply after the equivalence point because of the presence of excess Na+ and OH-. The plot becomes steeper than in the previous phase because the OH- are not neutralized and their mobility is higher than that of acetate ions. CH3COOH + NaOH→ CH3COO- + Na+ + H2O CH3COOH CH3COO- + H+ The intersection of the linear sections of the graph is often not clearly visible, and the measurement is therefore not precise. This problem can be avoided by using the method described in Section 3. 5 3. Titration of a weak acid with a weak base As an example, oxalic acid may be titrated with N-Methylglucamine (meglumine). Oxalic acid is a dicarboxylic acid. Its first proton (pKa1 = 1.05) can be titrated as a medium strength acid, while the second proton (pKa2 = 4.28) can be titrated as a weak acid. Nmethylglucamine, (C6H11O5·NH·CH3), a hexosamine, is used as standard solution; it contains a basic secondary amino group (pKa = 9.20) that can accept protons. The conductance of NMethylglucamine is negligible because its dissociation is very low. The first proton of oxalic acid influences the conductance in the early stages of the titration, because of its mobility. The mobile protons react with N-Methylglucamine and form N-Methylglucammonium ions which have strongly decreased mobility. That is why the conductance of the solution initially drops. The second proton of oxalic acid reacts according to the following equation: HOOC·COO- + C6H11O5·NH·CH3 → -OOC·COO- + C6H11O5·NH 2 ·CH3 The number of charged particles increases, and the conductance is therefore also increases. The titration graph shows a decrease at the beginning until it reaches a minimum, after which it increases slowly. After the equivalence point, N-Methylglucamine becomes dominant in the solution. Its dissociation is practically zero and the dilution of the solution is negligible. The conductance reaches a plateau. The intersection of the linear sections of the graph is relatively clear, and the determination of the equivalence point is therefore easy. 6 CONDUCTOMETRIC TITRATION OF CARBOXYLIC ACIDS Background: Conductometric titrations are suitable for the analysis of reactions in which the ion concentration changes or in which the concentration is kept constant but the mobility of the ions changes. Acetylsalicylic acid and benzoic acid are weak organic acids. N-Methylglucamine, a weak base, is used as a standard solution for their analysis. The conductance decreases at the beginning of the titration, and then increases moderately. The conductance does not change or only negligibly after the equivalence point is reached. The conductance is plotted as a function of the volume of standard solution. The intersection of the two sections is clearly visible, and the equivalence point can be determined graphically. The conductance increases rapidly after the equivalence point if a strong base (NaOH) is used for the analysis. 7 ANALYSIS OF ACETYLSALICYLIC ACID Definition: Acetylsalicylic acid contains not less than 99.5 per cent and not more than the equivalent of 101.0 per cent of 2-(acetyloxy)benzoic acid, calculated with reference to the dried substance. Characters: A white, crystalline powder or colourless crystals, slightly soluble in water, freely soluble in ethanol. It melts at about 143°C (instantaneous method). Quantitative analysis: Weigh 0.1500 g acetyl salicylic acid. Prepare two independent samples! Use two 150 ml beakers. Dissolve them in 5 ml of methanol and then add 40 ml of distilled water. Put one of your samples on the magnetic stirrer and place a stir bar in the solution. Immerse the electrode of the conductometer into the solution and add as much water to reach the black mark on the electrode. Turn on the conductometer with the right button under the screen. On the left of the screen µS (micro siemens) mS (milli siemens) and °C signs are visible. The instrument shows the actual setting that should be changed to µS range. Titrate the sample by addition of 0.5 ml portions of 0.1 M N-methyl-glucamine (meglumine) standard solution. Record the conductance after each 0.5 ml. Continue the titration until a total 15.0 ml of titrant is added. Plot the conductance as a function of the volumes of the standard solution. Calculate the percentage of acetyl salicylic acid content of the sample. Enter the result with two-decimal precision. 1 ml of standard 0.1 M N-methyl-glucamine is equivalent to 18.016 mg acetyl salicylic acid. Repeat the titration with 0.1 M sodium hydroxide standard solution and evaluate the different titration curves. 1 ml of standard 0.1 M NaOH is equivalent to 18.016 mg acetyl salicylic acid. 8 ANALYSIS OF BENZOIC ACID Definition: Benzocaine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of ethyl 4-aminobenzoate, calculated with reference to the dried substance. Characters: A white, crystalline powder or colourless crystals, very slightly soluble in water, freely soluble in alcohol. Quantitative analysis: Weigh 0.1200 g benzoic acid. Prepare two independent samples! Use two 150 ml beakers. Dissolve them in 5 ml of methanol and then add 40 ml of distilled water. Put one of your samples on the magnetic stirrer and place a stir bar in the solution. Immerse the electrode of the conductometer into the solution and add as much water to reach the black mark on the electrode. Turn on the conductometer with the right button under the screen. On the left of the screen µS (micro siemens) mS (milli siemens) and °C signs are visible. The instrument shows the actual setting that should be changed to µS range. Titrate the sample by addition of 0.5 ml portions of 0.1 M meglumine standard solution. Record the conductance after each 0.5 ml. Continue the titration until a total 15.0 ml of titrant is added. Plot the conductance as a function of the volumes of the standard solution. Calculate percentage of benzioc acid content of the sample. Enter the result with two-decimal precision. 1 ml of standard.1 M meglumine is equivalent to 12.21 mg benzoic acid. Repeat the titration with 0.1 M sodium hydroxide standard solution and evaluate the different titration curves. 1 ml of standard 0.1 M NaOH is equivalent to 12.21 mg benzoic acid. Notes: The temperature of the sample is not needed to be monitored to correct the result with a temperature factor because the temperature is kept constant during the measurement. The instrument need not to be calibrated with solutions of known conductance as relative changes are being recorded. 9 POTENTIOMETRIC (pH-METRIC) TITRATIONS Visual observation of the end-point by using an acidbase indicator is simple and convenient, but it may cause several problems. Instrumental methods are being used in most of the quantitative analyses in the Pharmacopeia; for acidbase titrations, the measurement of pH can be a possible solution. Pontentiometry is an analytical method that is based on the measurement of electrode potential. The electrode potential of the indicator electrode immersed in the analyte is used to determine the concentration of the sample. It is possible to measure only the potential of a cell, which is the potential difference between two electrodes. It is universally agreed that an arbitrary electromotive force (emf) is assigned to one electrode, and the potential of the second electrode can be measured. Two types of galvanic cells can be distinguished: a cell without transmission: Ag / AgCl / ZnCl2(c1) / Zn, and a cell with transmission: Ag / AgCl/ KCl(c2) // ZnCl2(c1) / Zn The major difference between the two cells is that there is an electrodeliquid interface in a cell without transmission, while there is a liquidliquid interface in a cell with transmission, where KCl and ZnCl2 solutions are in contact. The potential difference here is called the liquidliquid interface potential or diffusion potential. The potential of the cells includes the diffusion potential in every liquidliquid interface cell. The value of the diffusion potential can be decreased by using a salt bridge. A salt bridge consists of a concentrated or saturated solution of a specific salt, where the mobilities of its anions and cations are nearly the same. Potassium chloride (KCl) is most frequently used for this purpose, but when Cl- ions disturb the analysis, potassium or ammonium nitrate (KNO3 or NH4NO3) is used. A salt bridge actually means the insertion of two diffusion potentials. As the electrolyte concentration of the salt bridge is much higher than the analyte concentration, the two diffusion potentials are influenced by the K+ and Cl- or NH4+ and Clions. The mobilities of these ions are nearly the same, the value of the diffusion potential is low, and the potentials at the two interfaces are also really close to each other. The value of the diffusion potential will be very low; however, it cannot be eliminated completely, but only decreased to a minimum. The electrode potentials agreed by convention are determined by comparison with the standard hydrogen electrode. The reference electrode, the standard hydrogen electrode, is set to 0.00 V. The standard hydrogen electrode is a platinized platinum electrode that is immersed in 1.0 mol/dm3 HCl and pure hydrogen gas (H2) is bubbled through it. The pressure of the H2 is 0.1 MPa. Any electrode for which the electrode potential is not yet known, can be coupled with the standard hydrogen electrode to form a galvanic cell, and the potential of the galvanic cell gives the the potential of the unknown electrode. The table below shows several specific electrode potential values. The electrode potential can be positive or negative. A negative electrode potential means that the electrode is rather reducing relative to H+, while a positive value indicates a stronger oxidizing property than that of hydrogen. 10 Types of electrodes in potentiometry: 1. Electrodes working on the basis of equilibrium reactions (e.g. primary electrodes, secondary electrodes and redox electrodes) 2. Ionselective electrodes (membrane electrodes) (e.g. pHselective glass electrodes, metal ion selective glass electrodes and liquid membrane electrodes) 3. Moleculeselective electrodes (e.g. enzyme electrodes and gas moleculeselective electrodes) The pHsensitive glass electrode is the most important in the pharmaceutical analytical practicals. The most important part of the glass electrode that is used for pH measurement is a thin bulbform membrane made of hydrogensensitive glass attached to a nonhydrogenselective glass tube. The resistances of both the tube and the membrane are high. The H+ response is given only by this special glass membrane. The depth of immersion does not influence the measurement if the membrane is fully covered by the solution. Table of standard electrode potentials Li+(aq) + e- → Li(s) -3.04 IO-(aq) + H2O(l) + 2 e- → I-(aq) + 2 OH-(aq) 0.49 K+(aq) + e- → K(s) -2.92 Cu+(aq) + e- → Cu(s) 0.52 Ca2+(aq) + 2 e- → Ca(s) -2.76 I2(s) + 2 e- → 2 I-(aq) + - 0.54 - - - - Na (aq) + e → Na(s) -2.71 ClO2 (aq) + H2O(l) + 2 e → ClO (aq) + 2 OH (aq) 0.59 Mg2+(aq) + 2 e- → Mg(s) -2.38 Fe3+(aq) + e- → Fe2+(aq) 0.77 3+ - - 2+ Al (aq) + 3 e → Al(s) -1.66 Hg2 (aq) + 2 e → 2 Hg(l) 0.80 2H2O(l) + 2 e- → H2(g) + 2 OH-(aq) -0.83 Ag+(aq) + e- → Ag(s) 0.80 Zn2+(aq) + 2 e- → Zn(s) -0.76 Hg2+(aq) + 2 e- → Hg(l) 3+ - - 0.85 - - - Cr (aq) + 3 e → Cr(s) -0.74 ClO (aq) + H2O(l) + 2 e → Cl (aq) + 2 OH (aq) 0.90 Fe2+(aq) + 2 e- → Fe(s) -0.41 2Hg2+(aq) + 2 e- → Hg22+(aq) 0.90 Cd2+(aq) + 2 e- → Cd(s) -0.40 NO3-(aq) + 4 H+(aq) + 3 e- → NO(g) + 2 H2O(l) 0.96 2+ - - - Ni (aq) + 2 e → Ni(s) -0.23 Br2(l) + 2 e → 2 Br (aq) Sn2+(aq) + 2 e- → Sn(s) -0.14 O2(g) + 4 H+(aq) + 4 e- → 2 H2O(l) 2+ - 1.07 + 2- - 1.23 3+ Pb (aq) + 2 e → Pb(s) -0.13 Cr2O7 (aq) + 14 H (aq) + 6 e → 2 Cr (aq) + 7 H2O(l) 1.33 Fe3+(aq) + 3 e- → Fe(s) -0.04 Cl2(g) + 2 e- → 2 Cl-(aq) 1.36 2H+(aq) + 2 e- → H2(g) 0.00 Ce4+(aq) + e- → Ce3+(aq) 4+ - 2+ + - 1.44 - 2+ Sn (aq) + 2 e → Sn (aq) 0.15 MnO4 (aq) + 8 H (aq) + 5e → Mn (aq) + 4 H2O(l) 1.49 Cu2+(aq) + e- → Cu+(aq) 0.16 H2O2(aq) + 2 H+(aq) + 2 e- → 2 H2O(l) 1.78 ClO4-(aq) + H2O(l) + 2 e- → ClO3-(aq) + 2 OH-(aq) 0.17 Co3+(aq) + e- → Co2+(aq) 1.82 - - - 2- 2- AgCl(s) + e → Ag(s) + Cl (aq) 0.22 S2O8 (aq) + 2 e → 2 SO4 (aq) 2.01 Cu2+(aq) + 2 e- → Cu(s) 0.34 O3(g) + 2 H+(aq) + 2 e- → O2(g) + H2O(l) 2.07 - - - - ClO3 (aq) + H2O(l) + 2 e → ClO2 (aq) + 2 OH (aq) - 0.35 - F2(g) + 2 e → 2 F (aq) 11 2.87 The inner and outer surfaces of the membrane are hydrogensensitive. The electric potential at the outer surface, which depends on the proton concentration of the analyte, is usually measured with a secondary electrode, e.g. Hg-Hg2Cl2 or Ag-AgCl. There is a high buffer capacity reference solution inside the electrode where the reference electrode (usually AgAgCl) can be found. The schematic diagram of the whole electrochemical cell: Ag-AgCl internal Internal buffer electrode solution pH sensitive glass membrane Analyte solution Outer reference electrode (Hg-Hg2Cl2 (Single vertical lines indicate the phase borders, while the double vertical line denotes a salt bridge or diaphragm.) A combined glass electrode in which the reference electrode is inbuilt is used during the pharmaceutical analytical practicals. Shematic diagram of a glass electrode 12 The practice of potentiometric analysis: The measurement requires the following components: a solution of the analyte an indicator electrode (working (half) cell) a reference electrode (reference (half) cell) a potentiometer a closed circuit (salt bridge) Types of potentiometric analysis: direct potentiometry indirect potentiometry (potentiometric titration) The concentration of the electrode active material is calculated from the emf or the value of the electrode potential by using the Nernst-equation: E E0 R T ln(a ) zF where: a = activity (a = f c; f = activity coefficient; f 1, so a c in the case of dilute solutions) R = universal gas constant, 8.314 J/(mol K) T = absolute temperature (K) F = Faraday constant (96,487 C/mol) Introducing the constants: E E0 0.059 log(c) z where z = moles of electrons transferred in the cell reaction c = concentration The concentration of the electrodeactive sample can be calculated if the electrode potential is determined (known). Direct potentiometry is fast and easy to automatize, but there are limitations of its use because of the possible errors. Determination of the endpoint of a titration is also possible with potentiometric titration. The indicator electrode is immersed in the solution of the sample that contains the 13 electrodeactive material in this case and the emf is measured as a function of the volume of the standard solution. The titration curve is determined experimentally and its inflexion point indicates the equivalence point of the titration. The accuracy of the measurement depends on the determination of the endpoint of the titration and not on the accuracy of the measurement of the emf, and thus the error will be smaller. E 0.059 pH The use of indicator dyes is not necessary during the application of potentiometry, so any indicator error is eliminated. Potentiometric determination of the end-point is more sensitive than visual methods. It can be applied for solutions one order of magnitude more dilute than those where visual end-point determination is used. This method can be applied for all the titrations where either of the reactants can participate in a reversible electrochemical reaction for which a potentiometric electrode can be built. It is used for neutralization analysis, complexometry, argentometry and oxidimetry. Determination of the equivalence point graphically and numerically: The precision of the determination of the endpoint can be increased by the derivation of the titration curve. A local maximum or local minimum is visible in the first derivative of the curve; while the second derivative of the curve is zero. The electrode potential is directly proportional to the pH of the solution: E E0 R T log[ H ] zF 14 HOW TO USE THE GLASS ELECTRODE Combined glass electrodes are usually used when pH is monitored. The electrode must never be allowed to dry out. After use, the electrode must be rinsed with distilled water or, if it has been used in some non-aqueous medium, with other appropriate solution. In special cases, washing with water is not sufficient, and the electrode must be immersed in a cleaning solution for about 30-60 min, after which it must be rinsed and stored in an appropriate storage solution. The electrode must be detached from the main unit before it is turned off. The electrode must not be stored or turned upside down. The electrode is expensive. It must be immersed into the sample solution with extreme care, with the electrode kept about 4 cm beneath the surface. At the same time, it must be kept as far as possible from the magnetic stirrer. The pH-meter must be calibrated before use by immersing the electrode in different commercially available calibrating solutions. Various calibration points may be chosen for the calibration routine. However, calibration at two different pH values is used most frequently. During the calibration, the electrode is attached to the main unit, and is then rinsed with distilled water, dried and immersed in the chosen calibrating solution. The same measurement range is set and a waiting period is necessary until the pH value on the screen has been stabilized. The value is then recorded and stored in the memory unit of the pH-meter. After calibration, the electrode must be rinsed again with distilled water, and the analysis can then start. The calibration range must be chosen so that the measured pH or potentials fall into this range. The most frequently used calibration solution is pH 7.01 buffer solution. POTENTIOMETRIC TITRATION The combined glass electrode is immersed into the sample solution, the magnetic stirrer is set up for slow mixing and small quantities of standard solution are added. It is necessary to wait for a few seconds after the addition of each volume to allow the pH to stabilize. The pH values are recorded and plotted as a function of the volume of standard solution used. In acidbase titrations, the temperature may rise in case of because of the neutralizing heat. The pH may change if the temperature is not constant, and the sample flask must therefore be thermostated. EVALUATION OF THE MEASUREMENT The potentiometric titration can be evaluated numerically or graphically. The numerical evaluation may be performed manually or by computer. The pH values are plotted as a function of the volume of standard solution used. The inflexion points should be determined. The easiest way to find inflexion points is the tangential method. 15 For appropriate analysis of the curves, very accurate graphs should be plotted. The basic requirement is multiple recordings of the pH changes around the inflexion points. It is therefore necessary to know the expected equivalence point. This is possible if the dissociation exponents and/or the equivalence ranges are known. The standard solution is usually added in Tangental method for evaluation of a potentiometric titration curve to the sample 0.5-ml quantities, but in the range close to equivalence the quantities should be smaller, e.g. 0.1-0.2 ml. The pH change is monitored continuously, and when it changes by more then 0.1 pH unit the added volume of standard solution should be decreased. The added volume of standard solution can be increased again if there are two equivalence points between the two inflexion points and the pH does not change significantly after the second inflexion point. In order to determine the equivalence point, parallel straight lines are fitted to the initial and final sections of the titration curve, and a third straight line is fitted to the linear points around the inflexion points of the curve. The mean V (cm3) value of the intersections gives the volume at the equivalence point. Before use, the pH-meter must be set with the help of the tutor. QUANTITATIVE ASSAYS BY TITRATION WITH ALKALINE SOLUTIONS Titration with NaOH or KOH is used for the quantitative assay of acidic substances in around 100 cases, in the Pharmacopoeia. More than 50% of these assays are potentiometric titrations. Organic substances are usually dissolved in alcohol because of their limitied solubility in water. Inorganic substances are tested in hydrophilic solutions. 16 DINATRII PHOSPHAS DIHYDRICUS DISODIUM HYDROGENPHOSPHATE DIHYDRATE Na2HPO4·2H2O Mr 178.0 Definition: Content: 98.0 per cent to 101.0 per cent (dried substance). Characters: Appearance: a white or almost white powder or colorless crystals. Solubility: soluble in water, practically insoluble in ethanol (96 per cent). Background: Potentiometric titration is used in the Pharmacopoeia to assay the hydrated and dehydrated forms of NaH2PO4 and KH2PO4. The samples must be dried before the analysis and the mass loss on drying must be taken into account. The different compounds can be measured together during the titration, which means when Na2HPO4 is analyzed, NaH2PO4 content can also be determined. Two-step titration curves are obtained in all cases during the analysis of these substances. An analytically accurate quantity (25.0 ml) of 1 M HCl is added at the beginning of the measurement, and the sample is then titrated with standard 1 M NaOH. The phosphates react with HCl to form orthophosphoric acid (H3PO4). The excess HCl and the H3PO4 are titrated to reach the first inflexion point (V1 ml), and (25 ml - V1 ml) is proportional to the concentration of HPO42-. The first inflexion point can be calculated by using the dissociation exponents: ½(pKs1+pKs2); its value is ~4.6. Frequent measurement intervals should therefore be applied around the first inflexion point at pH 4.6. The second inflexion point is at pH ~9.7 (V2 ml), ½(pKs2+pKs3), when all the H2PO4- is converted to HPO42-. Frequent measurement intervals should again be recorded around the inflexion point and the titration should be continued until the pH changes is decreased dramatically (pH ~11). The result is calculated by using the equation below. Quantitative analysis: Disolve 2.0000 g of sample weighed with analytical accuracy in 50 ml of water R and add 25.0 ml of 1 M HCl. Titrate the sample potentiometrically to the first inflexion point (V1 ml) by using standard 1 M NaOH solution. Then continue the titration to the second inflexion point (total volume of 1 M NaOH solution required V2 ml). Calculate the percentage of Na2HPO4 by using the following formula: 1420 (25 f HCl V1 f NaOH ) m (100 d ) where d = percentage loss on drying. 17 Calculate the percentage Na2HPO4 contamination of the sample according to the following equation: V2 f NaOH 25 f HCl 25 f HCl V1 f NaOH This percentage content should not be greater than 0.025%. NATRII DIHYDROGENOPHOSPHAS DIHYDRICUS SODIUM DIHYDROGENPHOSPHATE DIHYDRATE NaH2PO4·2H2O Mr 156.0 Definition: Content: 98.0 per cent to 100.5 per cent (dried substance). Characters: Appearance: a white or almost white powder or colorless crystals. Solubility: very soluble in water, very slightly soluble in ethanol (96 per cent). Quantitative analysis Dissolve 2.5000 g of sample weighed with analytical accuracy in 40 ml water R. Titrate it with carbonate-free 1 M NaOH, determining the end-point potentiometrically. 1 ml of standard 1 M NaOH is equivalent to 0.120 g of NaH2PO4. Calculate the percentage NaH2PO4 content of the powder in a similar way as for Na2HPO4. 18 CHININI HYDROCHLORIDUM QUININE HYDROCHLORIDE C20H25ClN2O2·2H2O Mr 396.9 Definition: Content: 99.0 per cent to 101.0 per cent of alkaloid monohydrochlorides, expressed as (R)-[(2S,4S,5R)-5-ethenyl-l-azabicyclo[2.2.2]oct-2-yl]-(6-methoxyquinolin-4-yl)methanol]-hydrochloride (dried substance). Characters: Appearance: white or almost white or colorless, fine, silky needles, often in clusters. Solubility: soluble in water, freely soluble in ethanol (96 per cent). Background: The method described below is frequently specified for the analysis of organic amine salts amine hydrochlorides in the Pharmacopoiea. There are 78 such quantitative analyses, including papaverine·HCl, quinine·HCl, ephedrine·HCl and pseudo-ephedrine·HCl. The method is called displacement titration because the amine base is liberated from its hydrochloride form during the titration. Organic amine bases are not very soluble in water, and an alcoholic aqueous medium is therefore used. The acidic natures of the amine hydrochlorides differ, and the sample is therefore dissolved in ethanol and before the measurement 5 ml of 0.1 M HCl is added as an adjuvant solution. This HCl is not a volumetric solution; it must be added to the sample to allow the accurate determination of the first inflexion point, from where the amine hydrochloride is measured. In other words, at the beginning of the titration the excess HCl is measured by the addition of 0.1 M NaOH solution up to the first inflexion point. The volume relating to the second inflexion point depends on the quantity of the amine base. It is recommended to use smaller steps (e.g. 0.1-0.2 ml) around the inflexion points. If the pH jump is more than 0.1 unit, use 0.1 ml NaOH should be used as the amount added. The quantity of the amine base is proportional to the volume of NaOH added, which can be calculated by subtracting the volume added up to the first inflexion point that up to the second one. There are 19 special cases when diamine dihydrochlorides are tested (e.g. histamine·2HCl, meclozine·2HCl); in these titrations, three inflexion points can be observed. Standardization of the 0.1 M NaOH solution The procedure for the standardization of 0.1 M NaOH solution is similar to the displacement titration method: Accurately measure 0.1000 g of dried benzoic acid and dissolve it in 50 ml of alcohol. Add 5 ml of 0.1 M HCl and titrate the solution potentiometrically with 0.1 M NaOH. 1 ml of 0.1 M NaOH is equivalent to 12.21 mg of benzoic acid. Calculate the theoretical volume by using the equivalent mass of benzoic acid. Calculate the practical volume by subtracting the volume added up to the the first inflexion point from the volume added up to the second inflexion point. Calculate the factor of NaOH by using the following equation: f Vtheoretical V practical The benzoic acid should be very pure for the standardization. If only inpure benzoic acid is available, it should be purified in an appropriate sublimation apparatus. Quantitative analysis: Dissolve 0.2500 g of accurately weighed sample in 50 ml of alcohol R and add 5 ml 0.1 M HCl. Titrate the sample with 0.1 M NaOH, determining the end-point potentiometrically. Read the volume added between the 2 inflexion points. N.B. standard 0.1 M NaOH solution is made by the dilution of 1 M NaOH stock solution. NaOH solutions should always be standardized because NaOH pellets are hygroscopic and adsorb carbon dioxide (CO2). The standardization of standard NaOH solution is described in detail in the theoretical guidelines. 1 ml of 0.1 M NaOH is equivalent to 36.09 mg of C20H25ClN2O2·2H2O. Calculate the C20H25ClN2O2·2H2O percentage of the powder. 20 UNGUENTUM AD VULNERA (UNG. AD VULNER.) DERMATOLOGICUM. ANTISEPTICUM. Composition: Acidum salicylicum 0.6 g Vaselinum acidi borici ad 30.0 g Background: At the beginning of the titration, standard 0.1 M NaOH solution is added in 0.2-ml portions to the sample. The pH is recorded after each 0.2 ml. The pH may decrease and then slowly increase during the titration. The change is more dramatic around the equivalence point of the salicylic acid, after which the rate of pH increase slows down. After the equivalence point has been reached, the sample is overtitrated by the addition of at least 5 ml of 0.1 M NaOH solution in 0.5-ml portions and 2.0 g of mannitol is then added. Mannitol forms a complex with boric acid (H3BO3) that can be titrated as a stronger monoprotic acid than H3BO3 itself. The pH of the solution drops by 2-3 units. OH OH HO HO + B OH HO OH H NaOH O O + B HO O Na + H2O O boric acid - mannitol complex The titration is carried oot to reach the equivalence point of H3BO3 and continued with 5-6 ml of standard solution after the potential jump so as to be able to evaluate the result graphically. The pH is plotted as a function of the volume of standard 0.1 M NaOH solution. Two inflexion points are visible in the curve. The first is directly proportional to the amount of salicylic acid. The difference between the second and first equivalence points is directly proportional to the amount of H3BO3. The amount of salicylic acid and H3BO3 should be calculated in 30.0 g of sample. Quantitative analysis: An HI 9321 type pH-meter and an HI 1331 type combined glass electrode are used during the potentiometric measurements. The instrument can be connected to the mains through 21 an adaptor. Between measurements, the electrode is stored in a special storage solution that can be found in the cap of the electrode. Before the measurement is started, this cap should be removed and the electrode must be rinsed with water R before use. The lid should be kept in such a way as to keep the storage solution intact. The electrode must be connected to the instrument before the pH meter is turned on with the ON/OFF button. The electrode must be immersed into the sample solution so that a distance of at least 4 cm is kept between the bottom of the electrode and the surface of the solution. The monitor of the pH-meter shows the actual pH (it is necessary to wait a few seconds to let the value stabilize). Heat 1.4000 g of sample weighed with analytical accuracy with of 50.0 ml water to 100 °C, then shake it to dissolve the active ingredients, cool it down to room temperature and filter it to remove the base of the ointment. Put the sample solution (in a 100.0 ml beaker) on a magnetic stirrer and place a clean stir bar into the solution. Set the stirring speed to medium. The sample solution is titrated with 0.1 M NaOH. It is recommended to use smaller quantities at the beginning of the titration, e.g. 0.2 ml. Check and record the pH after the addition of each quantity of NaOH (wait a few seconds after the addition of NaOH to let the pH stabilize). During the titration, the pH first decreases slightly and increases slowly. Around the equivalence point of salicylic acid, the pH rises rapidly, and after the equivalence point it reaches a plateau. Continue the titration with 5-6 ml of additional 0.1 M NaOH after the equivalence point of salicylic acid has been reached. Then add 2.0 g of mannitol to the solution. Mannitol forms a complex with H3BO3 and this complex is a stronger acid than H3BO3 itself. At this point, the pH of the solution drops by 2-3 units. Continue the titration with 0.1 M NaOH to reach the next potential jump at the equivalence point of H3BO3; it is necessary to overtitrate so that graphical determination of the equivalence points is possible. Plot the pH values as a function of the volume of 0.1 M NaOH added. A curve with two potential jumps gives the volumes of NaOH needed to titrate salicylic acid and H3BO3, respectively. The volume added up to the first potential jump is equivalent to the amount of the salicylic acid, and the difference between the second and first potential jumps is equivalent to the amount of the H3BO3. Calculate the salicylic acid and H3BO3 contents of a 30.0 g sample. Give the results in grams with four-decimal precision. 1 ml of standard 0.1 M NaOH solution is equivalent to 13.812 mg of salicylic acid (C7H6O3). 1 ml of standard 0.1 M NaOH solution is equivalent to 6.183 mg of H3BO3. 22 SPECTROPHOTOMETRY Energy is absorbed by all atoms and compounds depending on their chemical structure. The structure of the molecule determines the interaction of the molecule and the electromagnetic radiation. The electromagnetic radiation absorbed is directly proportional to the concentration of the sample, and this phenomenon can therefore be used for analytical purposes. The method is simple, fast, sensitive, and specific, and is frequently used in analytical chemistry for quantitative determinations. The interpretation of absorption phenomena that occur in ultraviolet (UV) and visible (VIS) light is at the main focus of the pharmaceutical analysis practicals. The excitation of single σ-bonds in a molecule is very difficult; it may be achieved when far-UV light is used. Nonbonding electrons (n-electrons) in the outer shell (that are not involved in chemical bond formation) can be excited by UV light. π-electrons (double or triple bonds) can be excited by both UV and VIS light. The chromophore group of a molecule is responsible for its light absorption. Most chromophore groups contain one or more unsaturated bonds. A group of atoms attached to a chromophore which is able to modify how the chromophore absorbs light is called an auxochrome. The absorption maximum of a molecule can be influenced in the following ways: A bathochromic effect occurs when the absorption maximum shifts to longer wavelengths. The opposite is a hypsochromic shift, when the absorption maximum shifts toward shorter wavelengths. Hyperchromicity is the increase in absorbance of a material, while hypochromicity is the decrease in absorbance of the substance. These phenomena are used in practice when chromophore groups are built into a molecule: 23 Nitrobenzene is a typical chromophore; the nitro group of the aromatic ring intensifies the conjugation. Another similar molecule is trinitrophenol (picric acid), a yellow compound; its salts are called picrates. The measurement of steroids in the UV-VIS range on the basis of own light absorption at short wavelengths is really difficult. However when steroid derivatives are used on the basis of the following reactions: the analysis can be performed: When an aldehyde reacts with a primary amine, a Schiff base is formed. In the case of an aromatic amine, the conjugation of the molecule is extended. A bathochromic shift occurs. 24 The determination of protein concentration is possible in the UV range at a wavelength of 280 nm, when the absorption of the aromatic side-chains of phenylalanine, tyrosine and tryptophan is maximal. Complex formation is often used in practice, when the protein concentration is measured in the VIS range: Most such measurements are based on the fact that the peptide bonds of proteins are able to reduce copper ions (Cu2+) in alkaline media. The extent of the reaction is directly proportional to the amount of protein in the sample. The reduced copper ions (Cu+) form a colored product with a chelating agent, e.g. the BCA assay shown above. 25 The ninhydrin reaction is used to detect peptides/proteins. Ninhydrin can react with primary and secondary (noncyclic) amines. The product of the reaction is a conjugated Schiff base with an absorption maximum in VIS range. Since proline is an imino acid, it does not react with ninhydrin. Ninhydrin reaction NH3 + CO2 +RCHO O O OH OH + H C R OH COOH H NH2 O O ninhydrin O O OH OH + NH3 + H OH O O O OH N O O blue complex, max = 570 nm Determination of the amino groups in amino acids, peptides and proteins The blood glucose level can normally vary between certain values. It is very important to monitor it regularly to obtain acurate information about the carbohydrate metabolism of the body, and particularly to identify diabetes. Two basic techniques are used to determine the blood glucose level. The first is chemical method, in which the nonspecific reducing property of glucose is used. The concentration of glucose is revealed by the color and its intensity of an indicator. 26 1. Glucose + alkaline copper(II)tartrate copper(I)-oxide 2. Cu2+ + phosphomolybdic acid “Mo2O5” phosphomolybdic oxide (blue-colored endproduct) The colored end-product of the reaction can be measured by spectrophotometry. The measurement may be influenced by other reducing substances present in the blood, e.g. higher values may be detected in uremic patients. These problems can be eliminated by using newly developed enzymatic techniques. The most frequently used enzymes are (a) glucose oxidase and (b) hexokinase. (a) D-glucose + O2 glucose oxidase D-glucono-δ-lacton + H2O2 Reducing agents, e.g ascorbic acid, bilirubin, gluthathione and certain drugs may interfere with the determination. The method is not suitable for the determination of the blood glucose level in urine. (b) glucose + ATP glucose-6-P + NADP hexokinase glucose-6-P-dehydrogenase glucose-6-P + ADP 6-phosphogluconate + NADPH + H+ The reduced NADP coenzyme is determined at 340 nm. Besides photometry, electrochemical methods, wih the use of biosensors, may be applied to determine blood glucose levels. Amperometric glucometers measure the current on the surface of a working electrode due to the chemical reaction, as a function of the working electrode potential. Several factors like temperature, hematocrit, drugs, etc., may interfere with the measurement. The amount of substance involved in the electrode reaction can be determined by means of Faraday’s law: the amount of substance released is proportional to the total electric charge passed through the cell in coulometric glucometers: 27 m M Q nF m = the mass of substance liberated at an electrode (g) M = the molar mass of the substance (g/mol) n = is the valency number of the ions of the substance (electrons transferred per ion) F = 96,500 (C/mol), the Faraday constant Q = the total electric charge passed through the cell (C) The measurement is precise only if the system detects the electrons released during the redox reaction of the analyte. The interfering reactions must therefore be blocked. Coulometric analysis is slightly influenced by environmental factors. The reactions on the biosensors: D-glucose + O2 H2O2 glucose oxidase D-glükono-δ-lactone + H2O2 platinum anode 2 H + + O 2 + 2 e- The Beer-Lambert law describes the relationship between the concentration (c) and the absorbance (A) of the sample; it is valid only for dilute solutions: A log Io 1 log l c I T Transmittance is the ratio of the transmitted light and the total incident light, usually expressed as a percentage. It is not often used in practice because its graph is hyperbolic. T I Io A = absorbance Io = intensity of the incident light I = intensity of the transmitted light T = transmittance ε = molar absorbance (absorbance of a 1 mol/dm3 concentration solution if l=1 cm ) l = path length c = concentration of the sample (mol/dm3) 28 The analysis of a single compound in a sample is most often required. The absorption maximum of the molecule should first be determined, after which the absorbances of the calibration solutions are measured. The absorbance is plotted as a function of the concentration and the concentration of the sample can then be calculated on the basis of the fitted straight line. Multiple components in the same sample can also be analyzed. The choice of the appropriate wavelength is critical in this case. The best choice is a wavelength where only one of the molecules has an absorption maximum and the absorbances of the other molecules are close to zero. The absorbances of all the components in a sample are added: A = Acell + Asolvent + Areagents + Aunknown sample A’ = Acell + Asolvent + Areagents A’ = blank solution Aunknown sample = A – A’ The absorbance of the blank solution should be used to set the scale of the spectrophotometer to zero because there are colored reagents such as yellow iron(III) chloride (FeCl3) solution. Double-beam spectrophotometers are used during the practicals of pharmaceutical analysis. A schematic diagram of the instrument is as follows: Light source Monochromator Sample cell Detector Reference cell Double-beam spectrophotometer I. A tungsten lamp is used as a light source in the VIS range. The lamp contains a straight filament. The emitted electromagnetic waves cross the wall of the lamp perpendicularly. Light sources in the UV range are the mercury-vapor lamp, the hydrogen lamp and the deuterium lamp. The deuterium lamps used in modern spectrophotometers cover the UV range completely. The mercury-vapor lamp is official in Ph.Hg.VIII. The disadvantage of the mercury-vapor lamp relative to the deuterium lamp is that it has an energy minimum at ~200 nm and it cannot be used in this wavelength region. The lamps used in spectrophotometry are filled with special gas. Because of the high pressure inside them, the glass can cause serious injuries if they are broken so they cannot be disposed of as communal waste. Spectrophotometers should be turned on 10-15 minutes before use. Prisms, half-prisms or diffraction gratings are used as monochromators in spectrophotometers. 29 Reference cell Aperture V-shaped mirror Light source Sample cell Detector Beam splitter Double-beam spectrophotometer II. Photocells that are especially sensitive for the electromagnetic radiation in the UV, VIS and near IR range can be used as detectors in spectrophotometers. Incident photons excite electrons and these free electrons fly from the cathode toward the anode when electromagnetic waves reach the surface of the photocell. The photocurrent depends on the intensity and wavelength of the exciting electromagnetic radiation. Semiconductors are more effective than photocells and they operate in a broad wavelength region. CCD (charge-coupled device) detectors are combined with photodiodes that transform light into an electronic signal. CCDs comprise major technology in digital imaging such as in photography. In photodiode array detectors, hundreds or thousands (e.g. 256, 512, 1024 or 2048) of photodiodes are used. Photodiode array detectors can be used in nanometer resolution. 30 Spectroscopic methods can be distinguished by the energy of the electromagnetic radiation: Name Gamma-rays X-rays Wavelength Effect Practical use 0.5-10 pm Excitation of nuclei Material sciences, synchrotrons. 0.01-10 nm Excitation of inner shell electrons Structure determination: X-ray diffraction Diagnostics UV light 10-380 nm Excitation of outer shell electrons Analytical chemistry: spectrophotometry. VIS light 380-780 nm Excitation of outer shell electrons Analytical chemistry: spectrophotometry. 780-2500 nm Excitation of vibrational and rotational states of molecules IR radiation 2.5-300 μm Excitation of vibrational and rotational states of molecules IR spectroscopy: identification tests Microwaves 0.3 mm - 1 m Electron spin excitation of spin transition, excitation of rotational states of molecules ESR = Electron spin resonance spectroscopy Radiowaves 1-300 m Excitation of nuclear spins NMR = Nuclear magnetic resonance spectroscopy: structure and qualitative analysis Near-IR 31 Near-IR spectroscopy: Quality control, identification of products based on pigment dyes 32 PULVIS CHINACISALIS CUM VITAMINO C (PULV. CHINACISAL. C. VIT. C) ANTIPYRETICUM. ANALGETICUM. Components: Chinini sulfas 0.15 g Acidum ascorbicum 1.50 g Acidum acetylsalicylicum 6.00 g For 10 doses of divided powder Background: The acetyl group in acetylsalicylic acid can be removed by alkaline hydrolysis. The hydrolysis is faster at higher temperature. 33 In acidic media, salicylic acid forms a violet complex with Fe3+ that can be analyzed in the visible range. I. Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of acetylsalicylic acid: Dissolve 0.1000 g of acetylsalicylic acid R (pure reference material) weighed with analytical accuracy in 10 ml alcohol and then add 1.25 ml of freshly prepared 10% aqueous KOH solution. Five min later, add 0.25 ml 25% HCl to the solution and then dilute it to 100.0 ml with water in a volumetric flask. The acetylsalicylic acid concentration of this stock solution is 1 mg/ml or 1000 µg/ml. Use this stock solution to make a 10-fold diluted solution A: mix 10.0 ml of the stock solution with 5.0 ml of 1% FeCl3 solution made with 0.1 M HCl (solution I) and dilute it to 100.0 ml with water in a volumetric flask. The concentration of solution A is 100 µg/ml. Use solution A to prepare the solutions needed for the calibration curve. Make 20, 40, 60 and 80 µg/ml solutions in 25-ml volumetric flasks. Measure the appropriate amounts from solution A into 25-ml flasks and make up to volume with the reference FeCl3 solution (solution II: 5.00 ml of 1% FeCl3 made with 0.1 M HCl and diluted to 100.0 ml with water) to keep the FeCl3 concentration constant (0.05%). The 100 µg/ml solution is solution A itself. (If the weight of salicylic acid is different from 0.1000 g, the concentrations will be different, and that must be taken into account during the calculation.) Amount of solution A to prepare: Absorbance 20 µg/ml solution ml 40 µg/ml solution ml 60 µg/ml solution ml 80 µg/ml solution ml Determine the absorption maximum of acetylsalicylic acid according to the manual of the spectrophotometer between wavelengths of 500 and 600 nm. (The 60 µg/ml calibration solution should be used.) Measure the absorbances of the calibration solutions at the absorption maximum of the acetylsalicylic acid. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its acetylsalicylic acid concentration: Dissolve 80 mg of substance in 10.0 ml of alcohol then add 1.25 ml of freshly prepared 10% KOH solution. Five min later, add 0.25 ml of 25% HCl and then dilute it up to 100.0 ml with water. Mix 10.0 ml of this solution with 5.0 ml of 1% FeCl3 solution made with 0.1 M HCl (solution I) and dilute to 100.0 ml with water. Calculate the acetylsalicylic acid content of the powder, using the concentration calculated by the spectrophotometer on the basis of the calibration curve. Determine the specific absorbance of the acetylsalicylic acid too. 34 Background: The concentrations of multiple components in a sample can be analyzed by spectrophotometry. Measurements can be made when a wavelength can be found where one compound has an absorption maximum and the absorbances of the other compounds are zero. Appropriate wavelengths can be found for two-component samples, but the probability decreases when three- or multiple-component samples are to be analyzed. The amounts of both acetylsalicylic acid and quinine sulfate can be determined by spectrophotometry in Pulivis chinacisalis. Acetylsalicylic acid absorbs UV light, but its absorption maximum is shifted toward the VIS range (bathochromic effect) as a result of complex formation, and therefore quinine sulfate does not influence the measurement. The absorbance changes rapidly if a peak is sharp, whereas the change is not so dramatic if peak is broad. The shape of the absorption maximum peak should be considered during the measurement. Choosing a broad peak is more appropriate, as indicated in the figure above. The measurement wavelength chosen for the analysis is closer to the absorption maximum in the case of a broad peak. II. Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of quinine sulfate: Dissolve 50.0 mg of accurately pure quinine sulfate R weighed with analytical accuracy with gentle heating in a mixture of 1.0 ml 0.05 M H2SO4 and 10.0 ml of alcohol. Cool the solution to room temperature and dilute it to 50.0 ml with water. The quinine sulfate concentration of this solution is 1.0 mg/ml. Prepare a 10-fold dilution in a 50.00-ml volumeric flask with the solution that contains 2.50 ml of 0.05 M H2SO4 and 25.0 ml of alcohol per 250.00 ml. The concentration of the 10-fold diluted solution is 100 µg/ml. (If the weight of quinine sulfate is different, the concentration will be different and that must be taken into account.) Use 35 the 100 µg/ml solution to make the following calibration solutions in 25.0-ml volumeric flasks: 10.0, 20.0, 30.0, 40.0 and 50.0 µg/ml. Use the above-mentioned acidic alcoholic solution to make the dilutions. Amount of 100 µg/ml solution to prepare: Absorbance 10 µg/ml solution ml 20 µg/ml solution ml 30 µg/ml solution ml 40 µg/ml solution ml 50 µg/ml solution ml Use the 30 µg/ml calibration solution to determine the absorption maximum of quinine sulfate between wavelengths of 300 and 400 nm according to the manual of the spectrophotometer. When the absorption maxima are known, set the spectrophotometer to the longer wavelength and determine the absorbances of the calibration solutions. The reference solution is the acidic alcoholic solution used for the dilutions. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its quinine sulfate concentration: Dissolve 0.1500 g of sample weighed with analytical accuracy with gentle heating in the mixture of 1.0 ml of 0.05 M of H2SO4 and 10.0 ml of alcohol. Cool the solution to room temperature and dilute it to 100.0 ml with water. Measure the absorbance of the sample. Calculate the quinine sulfate content of the powder, using the concentration calculated by the spectrophotometer on the basis of the calibration curve. Determine the specific absorbance of the quinine sulfate too. N.B. The powder is made only for the pharmaceutical analysis practicals. The acetylsalicylic acid and quinine sulfate contents may be different from those in the original formulation. 36 TABLETTA ASPIRINI 500 (ASPIRIN TABLET 500) ANTIPYRETICUM. ANALGETICUM. Composition: Acidum acetilsalycilicum 500 mg Cellulosum (pulvis) qu. s. Amylum maydis qu. s. for each tablet Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of acetylsalicylic acid: Dissolve 0.1000 g of acetylsalicylic acid R weighed with analytical accuracy in 10 ml of alcohol and add 1.25 ml of freshly prepared 10% KOH. Five min later, add 0.25 ml 25% HCl to the solution and then dilute it to 100.0 ml with water in a volumetric flask. The acetylsalicylic acid concentration of this stock solution is 1 mg/ml or 1000 µg/ml. Use this stock solution to make a 10-fold diluted solution A: mix 10.0 ml of the stock solution with 5.0 ml of 1% FeCl3 made with 0.1 M HCl (solution I) and dilute it to 100.0 ml with water in a volumetric flask. The concentration of solution A is 100 µg/ml. Use solution A to prepare the solutions of the calibration curve. Make 20, 40, 60 and 80 µg/ml solutions in 25-ml volumetric flasks. Measure the appropriate amounts from solution A into 25-ml flasks and make them up to volume with the reference FeCl3 solution (solution II: 5.0 ml of 1% FeCl3 made with 0.1 M HCl and diluted to 100.0 ml with water) to keep the FeCl3 concentration constant (0.05%). The 100 µg/ml solution will be solution A itself. (If the weight of the salicylic acid differs from 0.1000 g, the concentrations will differ and this must be taken into account during the calculation.) Amount of solution A to prepare: Absorbance 20 µg/ml solution ml 40 µg/ml solution ml 60 µg/ml solution ml 80 µg/ml solution ml Determine the absorption maximum of acetylsalicylic acid between 500 and 600 nm according to the manual of the spectrophotometer. (The 60 µg/ml calibration solution should be used.) Measure the absorbances of the calibration solutions at the absorption maximum of the acetylsalicylic acid. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its acetylsalicylic acid concentration: Weigh an intact pill with analytical accuracy into a 100-150-ml beaker and add 20.0 ml of alcohol, and then add 10.0 ml freshly prepared 10% KOH solution. Cover the beaker with a watch glass and boil the sample for 5 min. Cool the sample to room temperature, add 1.0 ml of 25% HCl and dilute make the volume up to a 100.0 ml with water in a volumetric flask. Mix 37 1.00 ml of this solution with 5.0 ml of 1% FeCl3 made with 0.1 M HCl (solution I) and dilute it to 100.0 ml with water. Calculate the acetylsalicylic acid content of the powder, using the concentration calculated by the spectrophotometer on the basis of the calibration curve. Determine the specific absorbance of acetylsalicylic acid too. 38 SUPPOSITORIUM PARACETAMOLI 500 MG (SUPP. PARACET. 500 MG) ANTIPYRETICUM. ANALGETICUM. Composition: Paracetamolum 3.00 g Adeps solidus 50 : Butyrum cacao 7:3 qu. s. for 6 suppositories Background: The following factss should be borne in mind when measurements are made in the UV range. Solvents that absorb UV light cannot be used, e.g. benzene or toluene. Some solvents can absorb UV light at shorter wavelengths (e.g. ~180 nm), which disturbs the analysis. In measurements at 200-210 nm, the absorption is independent of the structure of the the molecule. The absorption is slightly dependent on the structure at ~250 nm, and therefore it is very important to note the absorption maxima. The absorption peak is definitely not specific for the analyte when the absorption maximum is at ~200 nm and the absorbance is higher than 1 (A>1). The measurement should be performed by setting the spectrophotometer to the absorption maximum of the second peak, e.g. at 280 nm in the figure above. 39 Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of paracetamol: Weigh 0.0500 g of paracetamol R with analytical accuracy, dissolve it in 5 ml of chloroform and add methanol to make it up to volume in a 50-ml volumetric flask (solution A). Dilute 1 ml of solution A with methanol to 50 ml (solution B) and use this solution to make 2.0, 4.0, 6.0, 8.0 and 10.0 µg/ml calibration solutions in 25-ml volumeric flasks. Amount of solution B to prepare Absorbance 2 µg/ml solution ml 4 µg/ml solution ml 6 µg/ml solution ml 8 µg/ml solution ml 10 µg/ml solution ml Determine the absorption maximum of paracetamol by using the 6.0 µg/ml calibration solution between 200 and 300 nm according to the manual of the spectrophotometer. When the absorption maxima are known, set the spectrophotometer to the longer wavelength and determine the absorbances of the calibration solutions. The reference solution is the acidic alcoholic solution used for the dilutions. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its paracetamol concentration: Weigh 0.3600 g of suppository, which contains ~60 mg of paracetamol, melt it on a hot plate and mix it with 10.0 ml of chloroform. Add methanol to make it up to volume in a 100-ml volumetric flask. Wait 20-30 min and then dilute 0.5 ml of the clear supernatant up to 50.0 ml with methanol. Measure the sample solution at the same wavelength and determine its concentration by using the calibration curve. Calculate the paracetamol content of a suppository of average weight. Calculate the specific absorbance of paracetamol. 40 SPARSORIUM ANTISUDORICUM (SPARS. ANTISUDOR.) DERMATOLOGICUM. ANTISUDORICUM. ADSTRINGENS. DESODORANS. Composition: Hexachlorophenum 0.60 g Acidum salicylicum 1.80 g Alumen 6.00 g Magnesii subcarbonas 20.00 g Zinci oxydum 20.00 g Talcum 20.00 g Background: Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of salicylic acid: Weigh 0.0500 g of salicylic acid R with analytical accuracy into a small beaker and dissolve it in a few ml of methanol and then wash the solution into a 50.0-ml volumetric flask with methanol and make it up to volume with the same solvent. Dilute 5 ml of the previous solution with solution II (5.00 ml of FeCl3 solution made with 1% 0.1 M HCl and come up to volume 100.0 ml with water) in another 50.0-ml volumeric flask. The salicylic acid concentration of this solution is 100 µg/ml. (If the weight of the salicylic acid is different, the concentration will be different.) Use this solution to prepare 10, 20, 30, 40 and 50 µg/ml calibration solutions in 25.0-ml volumetric flasks, using the same solution II for the dilutions. 41 Amount of 100 µg/ml solution to prepare Absorbance 10 µg/ml solution ml 20 µg/ml solution ml 30 µg/ml solution ml 40 µg/ml solution ml 50 µg/ml solution ml During the spectrophotometric measurements, use solution II as reference solution. Use the 30 µg/ml calibration solution to record the absorption curve according to the manual of the spectrophotometer and determine the absorption maximum of the curve. Measure the absorbances of the calibration solutions The sSpectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its salicylic acid concentration: Weigh 0.60 g of material, mix it vigorously with methanol and make the final volume up to 50.0 ml with the same solvent. Filter the suspension, throw away the first 10 ml and continue the filtration through the same filter paper into a clean beaker. Take 5 ml of the second filtrate to make a 10-fold diluted solution, using solution II (5.00 ml FeCl3 made with 1% 0.1 M HCl per and come up to volume 100.0 ml with water). Measure the sample solution an the same wavelength and determine its concentration by using the calibration curve. Calculate the salicylic acid content of the sample. Determine the specific absorbance of salicylic acid. 42 SOLUTIO METRONIDAZOLI (SOL. METRONIDAZ.) ANTIAPHTOSUM. Composition: Metronidazoleum 0.30 g Lidocainum 0.05 g Glycerinum 20.00 g Ethanolum 70% ad 30.00 g Preparation of the solutions of the calibration curve, determination of the absorption maximum of metronidazole: Weigh 0.0500 g of metronidazole R into a small beaker, dissolve it in methanol and wash the solution into a 50.0-ml volumeric flask (solution A). Prepare a 10-fold diluted solution: transfer 5.0 ml of solution A into a 50-ml volumetric flask and make it up to volume with methanol (solution B). The concentration of solution B is 0.100 mg/ml (100 µg/ml). (If the weight of the metronidazole differs from 0.0500 g, the concentration of the solution will differ and this should be taken into account.) Use solution B to make 2.0, 5.0, 10.0, 15.0 and 20.0 µg/ml calibrating solutions in 25-ml volumeric flasks. Amount of solution B to prepare Absorbance 2 µg/ml solution ml 5 µg/ml solution ml 10 µg/ml solution ml 15 µg/ml solution ml 20 µg/ml solution ml Determine the absorption maximum of metronidazole using the 10.0 µg/ml calibration solution, scanning the region between 300 and 400 nm according to the manual of the spectrophotometer. Measure the absorbances of the calibration solutions at the absorption maximum of metronidazole. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its metronidazole concentration: Weigh 0.5000 g of sample and dilute it to 50.0 ml with methanol. Transfer 5.00 ml of the solution to a 50.0-ml volumetric flask and make it up to volume with methanol. Measure the absorbance of the sample solution at the same wavelength and determine its concentration by using the calibration curve. Calculate the metronidazole content of the sample. Determine the specific absorbance of metronidazole. 43 PULVIS CHOLAGOGUS (PULV. CHOLAGOG.) CHOLAGOGUM. SPASMOLYTICUM. Composition: Homatropini methylbromidum 0.03 g Phenolphthaleinum 0.50 g Papaverini hydrochloridum 0.60 g Acidum dehydrocholicum 2.50 g Natrii salicylas 2.50 g Natrii bensoas 2.50 g For 10 doses of powder Background: Phenolphthalein turns colorless in acidic solutions and is pink in basic solutions. If the concentration of the indicator is particularly strong, it can appear purple. In very strongly basic solutions, the pink color of phenolphthalein fades and it becomes colorless again. Phenolphthalein is often used as an indicator in acidbase titrations. Its earlier use as a as laxative was stopped recently because its long-term application can cause malignancy. Phenolphthalein absorbs UV light in both its protonated and its deprotonated forms, due to its three aromatic rings. In the deprotonated form (slightly alkaline pH), the delocalization extends to the entire molecule. The excitation energy is decreased by the more extensive delocalization, and longer wavelengths (VIS) can therefore be used for its quantitative analysis. Phenolphthalein absorbs green light and transmits the complementary color pink (the one we see). The pH of the sample is shifted into the alkaline range by Na2CO3 during the analysis of phenolphthalein. 44 Barbiturates exhibit a similar phenomenon. The heterocyclic system of barbituric acid can be is stabilized in the oxo form at acidic pH, while the enol form is stable at alkaline pH. Barbituric acid shows both enol-oxo and lactam-lactim tautomerism. Barbiturates (5,5-disubstituted derivatives) shows only lactam-lactim tautomerism. Buffering of the sample solutions is therefore very important. However, possible decomposition reactions, e.g. acidic or alkalic hydrolysis must be avoided. The absorption spectrum changes as a function of pH. The molecule has different absorption maxima and minima at different pH values. The wavelength at which the absorbance is independent of the pH is called the isobestic point. This fact should be remembered during measurements in clinical chemistry. If the pH is set wrongly, the result will be false. Isobestic points of reference materials can be used to calibrate the spectrophotometer. Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of phenolphthalein: Weigh 0.0060 g of phenolphthalein R and dissolve it in methanol using a 100-ml volumetric flask (solution A). Take 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml portions of solution A and dilute them with 0.1 M Na2CO3 to 50.0 ml. 45 Concentration of the calibration solution Amount of solution A µg/ml solution 1 ml µg/ml solution 2 ml µg/ml solution 3 ml µg/ml solution 4 ml µg/ml solution 5 ml Absorbance The reference solution is 0.1 M Na2CO3. Determine the absorption maximum of phenolphthalein by using the 3rd calibration solution, scanning the region between 500 and 700 nm according to the manual of the spectrophotometer. Measure the absorbances of the calibration solutions at the absorption maximum of phenolphthalein. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its phenolphthalein concentration: Weigh 0.0300 g of sample and dissolve it in methanol in a 100.0-ml volumetric flask. Prepare two dilutions: take 4.0 ml and 8.0 ml in different 50.0-ml volumetric flasks and dilute them with 0.1 M Na2CO3. Measure the absorbances of the sample solutions at the same wavelength and determine their concentrations by using the calibration curve. Calculate the mass percent phenolphthalein content of the sample with four-decimal accuracy. Determine the specific absorbance of phenolphthalein. 46 DETERMINATION OF PROTEIN CONCENTRATION WITH THE BIURET REAGENT The proteins are macromolecules that have a large variety of functions and are essential for human life. They are built up from 20 amino acids, which are connected through peptide (amide) bonds. 50% of the dry material of a cell is protein. The total protein concentration in the human body is ~60-80 g/l. Background: Several methods are known for the determination of protein concentration. Some methods make use of the properties of peptide bonds or an amino acid side chains that react with certain reagents or dyes forming colored complexes. One well-known method is called the biuret reaction. The reagent, which contains Cu2+ ions in alkaline medium, forms bluishviolet complexes with peptide bonds. At least two peptide bonds are necessary for the reaction. Proteins fulfil this requirement in all cases. The following complex is formed: The biuret reagent contains CuSO4, NaOH, K,Na-tartrate and KI. (The K,Na-tartrate is necessary to keep Cu2+ in solution in the alkaline medium. KI stabilizes the reagent, so that the shelf life becomes longer.) Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of BSA: 20 mg/ml of protein stock solution made from BSA (bovine serum albumin) is used for the calibration solutions. Pipette 700 μl of biuret reagent into each of the test tubes and then add the following components according to the table: 47 Number of test tube Dist. water (ml) BSA stock solution (ml) 1 0.3 0 2 0.3 0 3 0.275 0.025 4 0.250 0.05 5 0.225 0.075 6 0.200 0.1 7 0.175 0.125 Concentration (mg/ml) Absorbance Incubate the reaction mixture tubes in a 35-40 C water bath for 10 min. Calculate the concentrations of the calibration solutions (column 4). 10 min later, use tube 5 to determine the absorption maximum in the range 500-700 nm. The reference solutions are in tube 1 and tube 2, which do not contain BSA. Measure the absorbances of the calibration solutions at the absorption maximum of BSA. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample and determination of its protein concentration: Take aliquots of 75 μl-t and 125 μl from the unknown sample into separate test tubes. Dilute them up to 300 μl with distilled water and then add 700 μl of biuret reagent. The measurement is more precise if the unknown sample is washed into a mixture of distilled water and the biuret reagent. It is recommended to pipette the reagent first, then add distilled water and finally wash the required amount of sample into the reaction mixture. Measure the absorbances of the samples. Calculate the protein concentration and give the final result in g/l with two-decimal accuray. Average the results on the individual samples. 48 ATOMIC ABSORPTION SPECTROMETRY Methods of sample preparation The following methods are used in the pharmaceutical industry to prepare samples for atomic absorption spectrometric measurements: dry ashing wet decomposition o in open vessels at atmospheric pressure o in closed Teflon beakers with steel covers, which allows the use of high pressure o with microwave radiation in closed plastic vessels: microwave-assisted sample preparation Microwave-assisted sample preparation will be described in detail here as this technique will be used during the practicals. The microwave-assisted wet decomposition of samples is becoming widely used in the pharmaceutical industry. This technique is suitable for the preparation of both solid and liquid samples. The procedure is carried out in a microwave oven. The inner wall of the oven is made of acid-proof steel covered with Teflon. The samples are fixed on a rotating plate in the microwave oven. These instruments have built-in pressure control units; the latest ones can also monitor the temperature. The sample vessels are made of acid-, temperature- and pressureproof plastic. The samples are weighed directly into the plastic vessels. 2–3 ml of liquid or 0.2–0.5 g solid sample is usually weighed for decomposition and concentrated H2SO4, HCl, HNO3, and/or H2O2 is then added. The membrane in the pressure valve should be checked. The vessels are then thightly sealed with the appropriate tool (torque wrench) and placed into a Kevlar jacket. The pressure valve opens only if overpressure develops. If the membranes are damaged, acid vapor is released and the samples are not suitable for pharmaceutical analysis, but the vessels remain unaffected. After the microwave treatment, the samples should be cooled to room temperature. During the process the carbon and hydrogen present in the organic components of the samples are turned into water and CO2 by the end of the wet decomposition; the inorganic compounds are dissolved in water or in the applied acids. The cooled samples are transferred into volumetric flasks for the analytical measurements, the vessels must also be rinsed out. Water molecules and other dipolar solvents are affected by the microwave treatment, making them oscillate with high frequency. This phenomenon heats the samples rapidly. The advantages of the microwave-assisted wet decomposition are: only small amount of reagents are necessary the closed system keeps the corrosive vapors inside the vessel only a short time is required for sample preparation Atomic spectroscopy Each element has a unique atomic electron configuration, and therefore a characteristic atomic spectrum. As each spectral line is sharp, the spectral lines of different elements do not or only rarely overlap. 49 Most elements vaporize at elevated temperatures and the molecules then dissociate into free atoms. The first step in atomic spectroscopy is the conversion of molecules into free atoms in the gaseous phase. If the atoms are then converted into an excited phase, they emit characteristic radiation when they relax. Analysis of the emitted radiation provides analytical information. These are the basic principles of the atomic spectroscopy. In other cases, the vaporization and atomization are not accompanied by excitation of the atoms, and the basis of the analytical measurements is the absorption of electromagnetic radiation by specific light sources. This latter technique is atomic absorption spectroscopy (AAS). Both methods are very effective, with detection limits in the ppm (parts per million) range, and the inductively coupled plasma (ICP) method (see below) is even suitable for the analysis of ppb (parts per billion) concentrations. As these are sensitive methods, problems arise from the aspect of accuracy. The methods are less accurate than, for instance, spectrophotometry. Atomic absorption spectroscopy AAS is useful for the analysis of more than 70 metals and semimetallic elements. Only a small amount of sample is usually used for the analysis. In the atomization unit of the instrument, the electrons of the atoms are promoted to higher orbitals by absorbing definite quantities of energy (e.g. in the cases of certain electromagnetic waves). The energy required for the excitation is specific for the electron transition of the element; a unique wavelength is specific for a certain element. The method is therefore selective. The sample is transformed into the gaseous phase and atomized during the analysis. Light passed through the atomized sample and is absorbed by the sample. The rate of absorption is directly proportional to the concentration of the analyte. Before the analysis, calibration solutions with known concentration must be prepared and the instrument must be calibrated. The major units of the instrument are the light source, the sample introduction unit, the atomization unit, the monochromator and the detector. The various spectrometers may differ in the atomization unit and the sample introduction unit. The methods available for sample analysis are flame, electrothermal and cold vapor-hydride atomization. Hollow cathode lamps are most commonly used as light sources in AAS. High voltage (100-400 V) is applied across the anode and cathode, resulting in ionization of the gas inside the sealed lamp. The emitted radiation is characteristic of this fill gas and the material of the cathode. The fill gas is usually Ar or Ne, depending on the analyte element. The spectral lines of the fill gas should not overlap with the analyte element. The cathode is made of the analyte element or covered with it. The electrons are accelerated between the cathode and the anode and ionize the fill gas, and the gas ions are therefore accelerated toward the cathode, causing the sputtering of atoms from the cathode. The atoms of the cathode become excited upon collisions with the fill gas and emit light as they fall back to the ground state. The emitted light is characteristic of the element. The intensity of the hollow cathode lamp can be influenced by the current. The atomization unit produces free atoms capable of the absorption of electromagnetic radiation. The atomization is either chemical or thermal; the latter is used for all elements except Hg. Three methods are available for thermal atomization: flame, electrothermal furnace (graphite tube) or radiofrequency-induced plasma. The sample is sprayed through the nebulizer into the flame, where the solvent is first evaporated rapidly. The molecules are atomized due 50 to the high temperature. A large amount of sample is usually required to obtain a continuos signal during the measurement. Premixed laminal gases are used in flame atomization. This type of flame is stable, the background is low and the transformations of the sample are separated throughout the height of the flame. Two types of gas mixes are used in most cases: acetyleneair and acetyleneN2O. Their temperatures differ; one is ~2000 K and is capable of atomizing most elements. The analytes should be volatile, e.g. Cl-, F- or H- salts. Other elements may form stable oxides at high temperatures, (e.g. Al, Ti, V, and W) and higher temperatures are necessary for their atomization; this is provided by acetyleneN2O (~3000 K). The atomization may be blocked by oxygen either in the flame or in the sample, resulting in the formation of oxides that dissociate only at very high temperatures. A reducing flame is therefore used, when the amount of fuel gas is higher than the oxidant gas. Flame atomization requires a large amount of sample, whereas in the case of a graphite furnace several µl of sample is sufficient and solid samples can also be analysed. The graphite furnace is heated up gradually to the appropriate temperature. The sample is first dried at ~400 K, and is next heated to 1000-1500 K to ash all the organic molecules. Finally, at 3000 K, the sample evaporates and atomizes. The gradual heating is necessary to avoid smoke in the light path. The graphite furnace serves as a cell. Graphite is an extremely good reducing agent at 2800-3200 K and therefore blocks the formation of metal oxides. The efficiency of the atomization is improved too. The sensitivity of this technique is 2–3 orders of magnitude higher than that of flame atomization. The method is sutable for the analysis of volatile samples. The emission spectra of hollow cathode lamps consist of multiple spectral lines due to the elements present: the material of the cathode, the fill gas (He or Ne) and the contaminants, if any. A monochromator system is therefore necessary for the selection of the appropriate wavelength. Littrow monochromators are usually used for this purpose. A spectral width of 0.2 nm is suitable for the AAS measurements. Narrowing the width of the slit is necessary until the spectral lines are well separated; there is no advantage of further narrowing. Photomultipliers are used for the detection of the intensity of the transmitted light. The current produced by the incident light is multiplied approximately 108-fold. The sensitivity and the stability of the photomultiplier are critical. The sensitivity depends on the photocathode material, while the stability is affected by the high-voltage electronic system. The magnitude of the high voltage influences the output of the photomultiplier. To be able to analyze multiple elements in the same sample, it was necessary to improve not only the light source, but also the detection methods. The photomultiplier was changed to a photodiode, a photodiode array, a CCD (charge-coupled device), or a CID (charge-injection device), so that simultaneous detection at multiple wavelengths became possible, the detection of inhomogeneity was solved, and the errors caused by the inhomogeneous dispersion of atoms were eliminated. Metal atoms absorb characteristic electromagnetic radiation, depending on their concentration. The detector measures the intensity of the transmitted light. The relationship between the absorbance and the concentration of the analyte follows the Beer-Lambert law: A lg Io l c I or A c 51 A number of elements in the same sample can be analyzed by AAS. The method is sensitive; it is capable of detecting as low as ppm concentrations, and is suitable for trace element analysis. ICP atom emission spectrophotometry The torch of the ICP (inductively coupled plasma) is heated to extremely high temperatures, and it is suitable for emission spectroscopic measurements as the samples are converted into an excited state in that temperature range. The ICP light source consists of three concentric quartz tubes in which different gases flow. The outer gas serves as a cooling gas, preventing the external quartz chamber from melting because of the high temperature (10,000 K). The quartz tubes are transparent and do not absorb the electromagnetic waves used during the analysis. The gas in the middle chamber elevates the plasma, while the gas in the middle of the light source delivers the sample into the plasma. Ar is usually used during the analysis. A coil of the radiofrequency (RF) generator surrounds part of this quartz torch. The Ar gas is ionized after the ignition of the torch. The RF generator creates an intense electromagnetic field that forces the charged particles to accelerate. A stable, high-temperature plasma of about 6,000-10,000 K is then generated as a result of the collisions between the neutral Ar atoms and the charged particles. The introduction of the samples depends on their state, their quantity, their physicochemical properties and the concentrations of the analytes. The solutions are simply sprayed by the nebulizer into the flame. Pneumatic, ultrasonic and high-pressure hydraulic nebulizers are usually used. V-shaped or Babington nebulizers are widely used, which are suitable for transferring solutions or suspensions without the risk of clogging capillaries 0.5-1.5 mm in diameter. The accuracy of the measurement is influenced by changes in the amount of charged particles during the analysis. The detection limits of the recently used instruments are below ppb for many elements, and they are therefore suitable for the analysis of biological samples containing low concentrations of metal ions. Uses of AAS and emission spectroscopy in the pharmaceutical industry: for the analysis of multivitamin complexes for the analysis of blood/serum, e.g. determination of Cu, As and Se for the analysis of medicines, e.g. the presence and amounts of reduction catalysts for the determination of contaminants for environmental analysis, e.g. Pb content determination 52 DETERMINATION OF MAGNESIUM CONTENT OF SPARSORIUM ANTISUDORICUM BY FLAME ATOMIC ABSORPTION Composition: Hexachlorophenum 0.60 g Acidum salicylicum 1.80 g Alumen 6.00 g Magnesii subcarbonas 20.00 g Zinci oxydum 20.00 g Talcum 20.00 g Preparation of the sample: Weigh 0.5000 g of sample into the container of the microwave destructor. Use a clean weighing boat. Add 20 ml of 4% H2SO4 to the sample. Wash the material off the wall. Set the container of the microwave destructor. Use the GYAK-HP500 menu to treat the sample. N.B. Be careful during the opening of the container after the microwave treatment because the sample might be very hot. Cool the sample and filter it. Wash the filtrate into a 100-ml volumetric flask and make it up to volume with water. Transfer 0.5 ml of the solution into a 50.0-ml volumetric flask and make it up to volume with 4% H2SO4 (solution A). Transfer portions of 5.0 ml and 10.0 ml from solution A into 100.0-ml volumetric flasks and make them up to volume with 4% H2SO4. Determination of the calibration curve and the magnesium concentration of the sample: Prepare calibration solutions with 0.20, 0.30, 0.40, 0.60 mg/l Mg concentration from 100 mg/l Mg solution. Transfer the aliquots into 25.0-ml volumetric flasks and make up the volume with 4% H2SO4. 100 mg/l Mg solution 0.20 mg/l 0.30 mg/l 0.40 mg/l 0.60 mg/l Measure the absorbances of the calibration solutions at 285.2 nm and 0.7 nm bandwidth and determine the calibration curve. Calculate the Mg content of the powder. 53 DETERMINTION OF MAGNESIUM CONTENT OF PULVIS NEUTRACIDUS BY FLAME ATOMIC ABSORPTION Composition: Calami rhizoma 1.0 g Frangulae cortex 1.0 g Natrii hydrogenocarbonas 8.0 g Bismuthi subnitras 14.0 g Magnesii subcarbonas 16.0 g For 40 divided powders Preparation of the sample: Weigh 0.2500 g of sample into the container of the microwave destructor. Use a clean weighing boat. Add 10 ml of 4M HNO3 to the sample. Wash the material off the wall. Set the container of the microwave destructor and place it into the instrument. Use the GYAK-HP500 menu to treat the sample. N.B. Be careful during the opening of the container after the microwave treatment because the sample might be very hot. Wash the sample into a 100.0-ml volumetric flask and make up the volume with water. Determination of the calibration curve and the magnesium concentration of the sample: Prepare a 500-fold diluted solution from the Mg stock solution in a 50.0-ml volumeric flask (solution A). Prepare the calibration solutions in 25.0-ml volumeric flasks from solution A. Make them up to the volume with 0.2 M HNO3. Prepare calribration solutions with Mg concentrations 0.25, 0.50, 0.8 and 1.00 mg/l. Amount of solution A 0.25 mg/l solution ml 0.50 mg/l solution ml 0.80 mg/l solution ml 1.00 mg/l solution ml Measure the absorbances of the calibration solutions at 285.2 nm and 0.7 nm bandwidth and determine the calibration curve. Calculate the Mg content of the powder. 54 DETERMINATION OF ACTIVE INGREDIENTS OF PANADOL EXTRA BY HPLC Compounds: Paracetamol 500 mg Caffeine 65 mg in each tablet Background HPLC (high-performance liquid chromatography), one of the most frequently used analytical techniques in the pharmaceutical industry, is suitable for the separation, identification and quantitation of the soluble components of a solution. The sample solution (an appropriate solvent is used) is injected onto a adsorbent layer (chromatographic column). The components of the sample adsorb on the surface of the column. The properties of the solvent and the compound influence the elution through the column. The eluted compounds are most often detected on the basis of their light absorption. The retention time is defined as the time when the compound appears at its maximal concentration in the chromatogram. The retention time depends on various factors, e.g. the analyzed substance, the chromatographic column, the solvent and the pressure. The HPLC separation of substances depends on the phenomena of absorption of the analyzed compounds and the chromatographic columns. The traditional normal-phase polar side-chains of silica gel columns adsorb polar molecules, particularly when an apolar solvent (moving phase) is used. Polar-polar interactions can therefore be strengthened by the application of an apolar solvent. Elution from the column (stationary phase) occurs as a function of the polarities of the molecules. Less polar compounds are eluted from the silica gel column earlier, as their interaction with the column is weaker. The elution can also be influenced by the composition of the solvent. In gradient elution, two solvents with different polarities are mixed and used as the moving phase. If the ratio of the solvents is changed, the adsorption interactions between the molecules and the column are also changed. At the beginning of the separation, a reverse polarity solvent is used, which promotes adsorption. The solubilities of polar compounds are lower in apolar solvents, and polar compounds preferably bind to the polar silica gel column. In contrast with the polar molecules, apolar molecules do not adsorb on the column, and their elution is faster. If the proportion of the more polar component of the solvent is increased, the polar molecules will also be eluted from the stationary phase. Most drug molecules are apolar, and rather hydrophobic, and reverse-phase chromatography is therefore used for their separation. In reverse-phase chromatography, alkyl/aryl groups are covalently attached to the traditional silica gel to form an apolar surface. Apolar compounds will bind to this with higher affinity, and polar ones with less or no affinity, and the polar compounds will therefore elute faster from the column. The current Pharmacopoeia accepts several reverse-phase columns. The names of the columns reflect the alkyl/aryl groups, e.g. octyl (C8), octadecyl (C18) or phenyl (C6H5). The type of the column, its length and diameter, and the particle size must be indicated when a chromatographic separation is performed. The composition of the solvent, the gradient, the applied pressure, the flow rate and the temperature are all important parameters of this separation technique. 55 Preparation of calibration solutions Two calibration stock solutions should be prepared. For Standard1Stock, weigh 100.0 mg of paracetamol and 13.0 mg of caffeine with analytical accuracy into a 50.0-ml volumetric flask. For Standard2Stock, weigh 105.0 mg of paracetamol and 15.0 mg of caffeine with analytical accuracy into another 50.0-ml volumetric flask. Dissolve the compounds in approximately 30 ml of solvent (90v/v% 50 mM phosphate buffer pH 6.3; 10 v/v% acetonitrile) then make the solutions up to volume with the same solvent. Record the accurate weights and concentrations. Dilutions: 1.5-ml aliquots of stock solutions are pipetted into separate 10.0-ml volumetric flasks and then made up to volume with the earlier solvent. After homogenization, pour the diluted Standard1 and Standard2 solutions into separate sample flasks. Calculate the concentrations of the stock solutions and the diluted standard solutions in mg/ml with fourdecimal accuracy, and record the results in the table below: Standard1 Paracetamol Weight (mg) Concentration of stock solution (mg/ml) Concentration of diluted standard solution (mg/ml) Caffeine Standard2 Paracetamol Caffeine Preparation of the sample Composite samples are analyzed, not individual samples, see below. So the goal is the determination of the content of the active ingredient, not uniformity analysis. Put therefore, e.g. 7 tablets, in the case of six students are attending the course, directly from the blister into a mortar. Grind the tablets in the mortar and homogenize the powder. Grinding is necessary because film tablets are used for the analysis and the grinding makes the active ingredients are more accessible for the solvents. Tare a 50-ml beaker on an analytical scale. Weigh 0.697 g ± 0.001 g of the powder. This is approximately the weight of one tablet. Record the accurate weight of the sample. Dissolve the active ingredients (paracetamol and caffeine) in a beaker with the earlier solvent (90 v/v% 50 mM phosphate buffer pH 6.3; 10 v/v% acetonitrile) by shaking the sample. After the shaking, pour the mixture into a 50.0-ml volumetric flask through a funnel, and then rinse the beaker with a small amount of solvent and finally make up the volume. The sample will be turbid, containing a flocculent precipitate, as not all of the components of the tablet are soluble in the solvent. After the larger particles have sedimented, pipette 300 µl of stock solution from the uppermost part of the solution into a 10.0-ml volumetric flask and make it up to volume with the solvent. Homogenize the sample solution. 56 The sample solution should be filtered before the HPLC measurement in order to remove the larger particles and contamination and to avoid clogging the instrument. Adjust the filter onto the 10-ml syringe (Millipore PVDF 0.45 µm). Pour the sample into the syringe and push it through the filter. Discard the first 7 ml. The remaining 3 ml is used for the measurement, and is filtered into the sample flask. The sample is ready for the analysis. 57 COMPLEXOMETRIC TITRATIONS During complexometric titrations, those standard solutions are used of compounds that form stable complexes with metal ions. A specific feature of the reaction is the formation of coordination complexes containing covalent bonds. The central metal ion is complexed by the ligands of the standard solution. Many metal ions can form six coordinate bonds, and therefore six electron-rich donor atoms are required for complex formation. The disodium salt of ethylenediaminetetraacetic acid (EDTA) is one the most commonly used standard solutions in complexometric titrations. EDTA has two acidic protons. Ethylenediaminetetraacetic acid disodium salt EDTA The complexes of EDTA contain a metal ions. The complex formation must be fast and quantitative if the reaction is to be used for volumetric analysis. The EDTA complexes are usually formed rapidly, and they are stable if the central metal ion is not a monovalent cation (e.g. an alkali metal ion), but their stability does depend on the pH of the solution. Buffer solutions are therefore always used during complexometric titrations in order to preserve the stability of the complex at the optimal pH. The stability of the complex also depends on the charge of the metal. The higher the elementary charge, the stronger the complex. Metal ions with an elementary charge 3 or 4 can be titrated in strongly 58 acidic medium (pH 2-3). During the titration of divalent transition metal cations, the pH should be kept between 5 and 6. Hexamethylenetetramine (methenamine or urotropine) is used to adjust the pH in this case. The complexes of divalent alkaline earth cations are the most sensitive to protonation, and they are therefore titrated at pH~10. NH4OH/NH4Cl buffer is suitable for this purpose. The pH~10 buffer pH≈10 is made in the following way: 50 g of NH4Cl is dissolved in 400 ml of 25% NH4OH solution, which is then made up to 1000 ml. 5 ml of this solution is added to 100 ml of sample. Approximately 2 g of solid urotropine is necessary to ensure pH 6. A standard disodium edetate solution can be prepared by direct weighing only if the material has first been dried at 80 °C for 1 h. Standardization depends on the application. Ca2+ or Zn2+ salts are usually used to determine the factor. H4EDTA is not hygroscopic, but its solubility in water is limited. The complexometric indicators are dyes that undergo color changes in the presence of certain metal ions. The end-point of the titration is reached when the metal has been completely displaced from the indicator; a constant color is therefore seen at the equivalence point. The complexometric indicators participate in protonation equilibrium processes and their colors depend on the pH of the solution. Eriochrome black T is used as indicator between pH 7 and the pH where the violet color of the metal ion complex turns blue at the equivalence point due to the presence of the single protonated blue form. (A double protonated red form exists at pH<6, but it is not not used as a complexometric indicator because its metal complexes are also red. Eriochrome black T is orange in its nonprotonated form at pH>12.) Some complexometric indicators are not stable in solution, and are therefore triturated with indifferent salts such as NaCl or KNO3 are usually used to make a solid mixture. The mixture usually contains 1% of the indicator and 0.1-0.2 g of it is used during the titration of a sample. ION INDICATOR pH END-POINT OF THE TITRATION FORM DISTURBING IONS Mg2+ Eriochrome black T 10 violetblue 1% (KNO3) Ca2+ Murexide >12 redviolet 1% (KNO3) Bi3+ Thymol blue 1-3 blueyellow 1% (KNO3) Fe3+ Hg2+ Thymol blue 6 blueyellow 1% (KNO3) Fe3+ Al3+ Dithizone 4 greenish-bluereddishviolet 0.025% (solution) 1-3 pinkyellow solution Ca2+ Calconcarboxylic acid >12 violetblue solution Bi3+ Xylenol orange 59 Al3+, Fe3+ O NH4 O R N R N N O O O N R N Murexide R S N N N H Dithizone 60 NH O 61 62 PULVIS NEUTRACIDUS (PULV. NEUTRACID.) ANTACIDUM. ADSTRINGENS. Composition Calami rhizoma pulvis : 1.0 g Frangulae cortex pulvis 1.0 g Natrii hydrogenocarbonas 8.0 g Bismuthi subnitras 14.0 g Magnesii subcarbonas 16.0 g Total mass: 40.0 g 40 doses of divided powder Preparation of the sample The sample needs special treatment before the analysis. The inorganic compounds in the sample are made ready for the analysis during this preparation. Certain components of the powder mixture, e.g. anthraquinone compounds in the frangulae cortex (glucofrangulins, frangulins or frangula emodin) form strong complexes with the metal ions in the sample, and would therefore disturb the analysis of the inorganic ions (Mg2+ and Bi3+). The sample must be heated to destroy its organic components. 63 Heating: 0.2500 g of sample is weighed with analytical accuracy into a porcelain jar. The sample is heated carefully and completely annealed. 10.0 ml of 30% HNO3 is added in small portions to the cooled sample until it has dissolved, and the solution is then washed it into a 100.0-ml volumetric flask with water. The Bi3+ and Mg2+ contents of the sample are analyzed by complexometry with sodium edetate standard solution in the presence of indicators. The analysis of these ions is possible in the same sample because the bismuth edetate complex is stable in acidic medium, whereas while the magnesium complex is stable in slightly basic solution. Determination of bismuth content: 20.00 ml of the stock solution is diluted to 100 ml in a 200-ml flask. Add 5-6 drops of xylenol orange indicator to the sample and titrate it with 0.01 M sodium edetate solution until the solution turns from pink to yellow. 1 ml of 0.01 M sodium edetate is equivalent to 2.09 mg of Bi. Determination of the magnesium content: Dilute 20.00 ml of stock solution with 70 ml of water in a 200-ml flask and then add 6 M NH4OH solution (about 2 ml) to neutralize the sample; it turns cloudy. Dissolve 0.25 g of NH4Cl in the sample solution; then mix it with 10 ml of 6 M NH4OH solution (pH~10). Add 0.1 g of Eriochrome black T and titrate it with 0.02 M sodium edetate standard solution until it turns from magenta to blue. The solution should keep its blue color for 5 min. 1 ml of standard 0.02 M sodium edetate is equivalent to 0.8064 mg of MgO. Notes: Examine the porcelain jar carefully before the experiment and look for fine cracks (cracked jars should not be used). Set the burner to jet lance, as otherwise the heating process is not successful. Distribute the powder evenly at the bottom of the pot. By the end of the heating, the sample is dark-red, but this changes to yellow when the sample cools down. The hot ceramic pot should not be put directly onto the bench, but also onto an asbestos surface. Mix the sample with a glass rod when it is being dissolved in HNO3 because this is a slow process. During the analysis of Bi, the indicator turns fro pink to yellow at the equivalence point when only a small drop is added. During the analysis of Mg the change in color of the indicator from magenta to blue is difficult to see. Use an overtitrated sample for comparison. The blue color should be stable for 5 min. 64 SUSPENSIO ZINCI AQUOSA (SUSP. ZINC. AQUOS.) DERMATOLOGICUM. Composition: Zinci oxydum 20.0 g Talcum 20.0 g Glycerinum 10.0 g Ethanolum 70% 10.0 g Aetheroleum menthae piperitae X gtt Solutio acidi borici 2% FoNo VII. 40.0 g Total mass: 100.0 g Determination of the zinc content of a sample The Zn content of a sample is determined by complexometric analysis. The suspension should be shaken very well before the measurement in order to analyze a homogeneous sample. Approximately 0.5 ml (0.500-0.600g) of suspension is used for the titration. The sample is transferred to a 50-ml beaker with a Pasteur pipette and its accurate weight is checked. 10 ml of R diluted acetic acid is added to the sample. The mixture should be shaken until it turns opalescent (about 1 min). Zn is dissolved in a weak acid, and free Zn2+ ions are obtained. Other components in the sample are not soluble in acetic acid, and therefore do not disturb the titration. The solution is washed into a 250-ml titration flask with 100 ml of R-water. 50 mg of xylenol orange trituration and 2 g of hexamethylenetetramine are then added to the sample. Immediately after the hexamethylenetetramine has completely dissolved, the pinkish-violet solution is titrated with 0.1 M sodium edetate standard solution until the solution turns yellow. Calculate the percentage ZnO content of the sample. Record the result with two-decimal precision. 1 ml of 0.1 M sodium edetate standard solution is equvivalent to 8.14 mg of ZnO. 65 ARGENTOMETRIC ANALYSIS Argentometric titration is a special type of volumetric analysis. The standard solution in argentometry is AgNO3, which can be stored for a long time when it is protected from light. The simplest method in argentometry is precipitation titration, when potassium thiocyanate (KSCN) (potassium rhodanide) or NH4SCN is used as a secondary standard solution. (Ph. Eur. recommends only standard NH4SCN solution.) Standard thiocyanate solutions slowly decompose, so they should be checked before use. As an example, during the determination of I- an excess of AgNO3 is added first to the sample, and then AgNO3 is titrated with KSCN. The SCN- ion forms a precipitate of AgSCN. If Fe3+ is added to the sample as an indicator, the red color of Fe(SCN)3 appears at the equivalence point of the titration (Volhard method). The proton of acidic N-H bonds can be replaced by Ag+ and non-ionic silver compounds are formed. Two types of measurements are possible in this case. When sulfadimidine is analyzed, direct titration is performed with standard AgNO3 solution and CrO4- as indicator. The silver compound of sulfadimidine is not ionic at the equivalence point, and the excess Ag+ forms a brown AgCrO4 precipitate with CrO4-. During the analysis of theobromine (Boie method), standard AgNO3 solution is added to the sample and the non-dissociating silver compound of theobromine and an equivalent amount of HNO3 are formed. The HNO3 is titrated with standard alkaline solution. This method is a combination of argentometry and acidimetry. The acidbase titrations can be followed potentiometrically, when the pH is checked. After the titration curve has been plotted, the equivalence point can be determined graphically. The first derivative curve should be used when the equivalence point is not sharp enough. Conductometry is a convenient method with which to follow precipitation titrations. 66 SPARSORIUM SULFABORICUM (SPARS. SULFABOR.) DERMATOLOGICUM. Composition: Sulfadimidinum 5.0 g Acidum boricum 5.0 g Total mass: 10.0 g CH3 O O N S N H N CH3 H2N Sulfadimidine Determination of the sulfadimidine content of a sample: Accurately weigh 0.3500 g of powder and dissolve it in acetone with gentle heating (it takes approximately 15 min). The dissolution of sulfadimidine in acetone is slow, and the sample must therefore be gently heated. It should not be boiled as acetone is flammable. 20 min is necessary for complete dissolution, and the solution is then cooled to room temperature. Next add 0.5 g of MgO and 2 drops of 10% K2CrO4 solution, when a yellow suspension is obtained. It is practical to use only a small amount of indicator (1-2 drops) because the color change can be observed more easily when light colors are used. Five min later, the reaction mixture is titrated with standard 0.1 M AgNO3 solution. The mixture is shaken thoroughly after each drop of standard solution. The titration should be carried out slowly, especially close to the end-point. It is recommended to wait 10-15 sec after the addition of each drop of standard solution. The end-point of the titration has not been reached if the solution turns back to yellow and the color of the precipitate is still white. Close to the end-point of the titration, it is recommended to wait a little after shaking. The yellow mixture turns salmon-pink at the end-point of the titration. 1 ml of standard 0.1 M AgNO3 is equivalent to 27.833 mg of sulfadimidine. Record the result in g with four-decimal precision for an average weight of 10 g. 67 REDOX TITRATIONS The application of redox reactions in volumetric analysis is called redox titration. We will discuss oxidimetry, when the standard solution is an oxidizing agent, while in case of reductometry the standard solution is a reducing agent. The most important oxidimetric methods: permanganometry (the standard solution is KMnO4) chromatometry (the standard solution is K2Cr2O7) cerimetry (the standard solution is Ce(SO4)2 or (NH4)2Ce(SO4)3) bromatometry (the standard solution is KBrO3) The most important reductometric method is ascorbinometry, where the standard solution is ascorbic acid. A special case of redox titration is iodometry, which can be used for either oxidimetry (iodimetry) or reductometry (iodometry). The standard solutions of iodometric methods are I2 and Na2S2O3 solutions. Bromatometry is sometimes combined with iodometry. After the addition of KBr and standard KBrO3 solution, excess Br2 is liberated. Br2 reacts with I- and I2 is formed. The I2 can be titrated with standard Na2S2O3 solution. The equivalence point of the redox titration can be detected by indicator dyes such as ferroin, by the reaction of I2 and starch, or simply through the disappearance of the color of I2. Redox titrations can be followed by potentiometry or biamperometry. The standard solution of bromatometry is KBrO3 solution, which can be prepared precisely by direct weighing. Standardization is not necessary as it is very stable. 0.033 M (0.1 N) and 0.02 M stock solutions are usually made directly; dilutions of stock solutions are also used. The standard solution of bromatometry can be used as a direct oxidizing agent or for the preparation of bromine solutions. Direct bromatometric analysis involves the titration of ascorbic acid with Br2 is formed when Br- ions are added to the reaction mixture, according to the following reaction: BrO3- + 5 Br- + 6 H+ = 3 Br2 + 3 H2O The Br2 formed reacts with ascorbic acid. The reaction is fast; dehydroascorbic acid is formed. 68 Back-titration is used when the reaction with Br2 is not fast enough for the analysis. Brand KBrO3 are added to the sample to form Br2. The reaction is allowed to go completion. The excess Br2 is titrated iodometrically. Organic compounds are brominated in addition or substitution reactions. Alkenes react with Br2 in addition reactions. Br2 is volatile, so special brominating flasks are used. Br Br2 Br This reaction is used for the analysis of unsaturated fatty acids. The iodine/bromine index gives information about the number of double bonds in the sample. The analysis of hexobarbital is also based on a Br2 addition reaction. Bromine substitution is specific for highly reactive aromatic compounds (containing first class substituents). Phenols, aminobenzenes, salicylic acid and anthranilic acid are analyzed in this way. In the analysis of salicylic acid by the Koppeschaar method, tribromophenol bromine is formed by bromine substitution and decarboxylation. OH OH CO2H Br Br + 3HBr +CO2 + 3Br2 Br 2,4,6-tribromophenol Salicylic acid O OH Br Br Br Br +Br2 ,-HBr +2HI, -I2-HBr Br Br Br Tribromophenol bromine 69 Tribromophenol bromine reacts similarly to elementary Br2 with I- ions; when the Br2 is titrated iodometrically, three bromines are involved in the reaction. Acetylsalicylic acid cannot be determined by bromination as it does not contain a free aromatic hydroxy group and it reacts slowly with Br2. When a longer reaction time is applied, the acetyl group is hydrolyzed and not acetylsalicylic acid, but salicylic acid is determined. Bromatometry is therefore not used for the analysis of acetylsalicylic acid. Several standard solutions are used in cerimetry. Ce(SO4)2, (NH4)2Ce(SO4)3 and Ce(NH4)2(NO3)6 standard solutions are all highly acidic. The hydrolysis of Ce salts is blocked when the pH is low. H2SO4 is usually used to decrease the pH, such cerimetric standard solutions being highly stable. Standard Ce(SO4)2 solution is prepared by dissolving Ce(SO4)2 in dilute H2SO4. Iodometry is used to standardize Ce(SO4)2 volumetric solutions. I2 is titrated when Na2S2O3 solution is used after the addition of KI. Standard (NH4)2Ce(SO4)3 solution is prepared by dissolving the compound in dilute H2SO4, and iodometry is used to standardize the solution. (NH4)2Ce(SO4)3 exists in two forms: anhydrous and hydrated; the anhydrous (NH4)2Ce(SO4)3 is used to prepare more accurate standard solutions. Ferroin indicator is used in cerimetry where three phenanthroline ligands coordinate an Fe or Fe3+ ion. 2+ Cerimetric oxidation is most often used for the measurement of Fe2+. The Fe content of Fe(II) gluconate is determined by the titration of standard (NH4)2Ce(SO4)3 solution in the presence of ferroin as indicator. The standard cerimetric solutions are strong oxidizing agents in aqueous medium. The following chemical equation describes the reaction: 4 Ce4+ + 2 H2O 4 Ce3+ + 2 “O··” + 4 H+ 70 Two Ce4+ ions as oxidizing agent are equivalent to one nascent oxigen “O··” (oxygen radical). This is a free radical reaction, where H2O accepts an electron, while Ce3+, a hydroxyl radical (HO·) and a proton are formed. Ce4+ + H2O Ce3+ + [H2O·]+ [H2O·]+ HO· + H+ 2 HO· H2O + “O··” The cerimetric determination of aminophenazone by the method of Rózsa is based on the above-mentioned reaction. Iodometric methods are divided into two different groups. The first type is when I2containing solutions are titrated with S2O32- (iodometry), and the second type is when reducing agents are titrated with standard I2 solution (iodimetry). Standard S2O32- solutions are made directly from powder, which is dissolved in freshly boiled distilled water (CO2 free) together with a small amount of (0.2 g) Na2CO3. I2 standardized with KBrO3 is used to determine the titer of the S2O32- solution. BrO3- + 6 I- + 6 H+ Br- + 3 H2O + 3 I2 S2O32- is one of the most unstable standard solutions. Its titer changes after preparation, first increasing slowly, and then decreasing gradually. It should therfore be stored for several days, when it reaches its final titer. If it is necessary to use the solution immediately after preparation, it must be standardized immediately before the titration. It must be borne in mind that this titer cannot be used in later measurements. The solution cannot be used for volumetric analysis if it contains colloidal sulfur precipitate. Such precipitation of sulfur is a result of 71 bacteria present in the solution. To prevent bacteria form growing in the solution, 1-2 ml of amyl alcohol is added to the volumetric solution before it is made sup to the final volume. I2 is not soluble in water; it is therefore dissolved together with KI, when KI3 is formed, a complex which dissolves very well in water (e.g. to prepare a 0.5 M standard solution, 127 g of I2 and 200 g of KI is dissolved and diluted to a final volume of 1000 ml) I2 + KI = KI3 (K = 710) Standard I2 solutions are kept in the dark and their titers are determined with Na2S2O3. Reducing agents can be analyzed with standard I2 solutions if they undergo oxidation instantaneously, and excess I2 is not necessary for the reaction. Standard KIO3 solution and back-titration of the excess I2 is used if the above requirements are not fulfilled. I2 is formed from KIO3 and I2- according to the following reaction: IO3- + 5 I- + 6 H+ = 3 I2 + 3 H2O I2 reacts with S2O32- to form tetrathionate ions (S4O62-): I2 + 2 S2O32- 2 I- + S4O62I3- + 2 S2O32- 3 I- + S4O62The amylose component of starch (water-soluble starch) is used as an indicator in iodometry. Amylose consists of 50-100 glucose monomers, which forms a spiral. I113- chains, consisting of 3 I3- + I2, enter these helices, and blue iodine–starch is formed. The color is so intense that it can be detected even at even I2 concentrations as low as 1·10-5 M. The solution is kept acidic when iodine–starch is used as indicator. Starch should be added just before the equivalence point of the titration because iodine–starch can precipitate and its reaction with S2O32- becomes very slow. The iodine–starch indicator does not function when the ethanol concentration is >50%. I- ions are necessary for the formation of iodine–starch. I2 is soluble in chlorinated hydrocarbons such as chloroform (CHCl3). Another typical indication method is when several ml of choloroform is added to the sample. Iodine dissolves in chloroform to give a purple color. This purple color disappears at the equivalence point of the titration. Vigorous shaking of the sample is necessary during the titration! Excess Br2 can be determined by iodometric titration with standard S2O32- solution after bromination reactions according to the following equation: Br2 + 2 I- I2 + 2 BrI2 + 2 S2O32- 2 I- + S4O62- 72 As an example, excess Br2 is determined after Br2 addition in the case of hexobarbital, or bromine substitution in the case of salicylic acid. I2 does not react with olefins or aromatic compounds, and indirect analysis therefore is used in these cases. Strong reducing agents can be titrated with standard I2 solution (iodimetry). The sulfur dioxide (SO2) liberated from noraminophenazone sodium salt by acidic hydrolysis is measured by standard I2 solution. SO32- + I2 + H2O = SO42- + 2 I- + 2 H+ Ascorbic acid can be titrated with I2 solution directly. Other redox techniques, such as permanganometry, chromatometry and ascorbinometry, are not discussed in detail here, as they are not used in the pharmacopoeia. Biamperometry (dead-stop titration) is especially useful for following redox titration reactions. Electric current flows while both the oxidized and reduced forms are present. The current stops at the equivalence point of the titration, when only either the oxidized or the reduced form is present (e.g. the reduced form is completely oxidized). When only one form is present at the beginning of the analysis, current flows only when the other form appears in the solution. The current therefore increases at the beginning of the titration, and then slowly decreases as the concentration of the first form decreases. A bell-shaped titration curve is observed. If the redox reaction of the analyzed sample is reversible electrochemically, e.g. Fe2+ is titrated cerimetrically, the current increases after the equivalence point is reached. The equivalence point is the minimum in the titration curve. Biamperometry is used for water analysis by the Fischer method. 73 SUPPOSITORIUM ANTIPYRETICUM PRO INFATE VEL PRO PARVULO (SUPP. ANTIPYRET.) ANTIPYRETICUM. ANALGETICUM. Composition: Aminophenazonum 0.45 or 0.9 g Adeps solidus compositus qu.s. 6 suppositories Background: During the quantitative analysis, the double bond of the pyrazolone ring is oxidized to a diacyl hydrazine compound (“dioxypyramidone”) by four Ce4+. The equivalent weight is therefore one-fourth of the molecular weight. Water is oxidized by Ce4+ ions as follows Ce4+ + H2O → [H2O]+ Ce3+ + [H2O]+ → 2 HO → HO + H+ H2O + O The pyrazolone ring is oxidized to dioxypyramidon by nascent oxygen (O). Aminophenazone is a strong carcinogenic agent because nitrosodimethylamine may be formed in the living body during its metabolism. 74 H3C O N N H3C Nitrosodimethylamine It is not registered in Ph.Eur. Determination of the aminophenazone content of a sample: Rózsa method: A suppository is melted in a beaker and 0.2-0.3 g of melted sample is weighed with analytical accuracy into a 100-ml titration flask. 10.0 ml of 15% of H2SO4 is added to the sample and the mixture is heated to about 40 °C to dissolve the aminophenazone. The sample is allowed to cool to room temperature, 15 ml of distilled water and 1 drop of ferroin indicator are added to the solution, and titrated with standard 0.1 N (0.1 M) Ce(SO4)2 solution. The orange color of the sample turns green at the end-point of the titration. The titration should be continuous without any disruption. The color of the sample may turn light-green before the end-point, but it may turn back to orange during intensive shaking. The end-point may then be reached by the addition of 1-2 more drops of standard solution. The green color should persist for half a minute. Parallel titrations should be performed, because the sample may not be homogeneous. 1.00 ml of standard 0.05 M Ce(SO4)2 solution is equivalent to 2.891 mg of aminophenazone. The final result should be calculated in mg with two-decimal precision. The average weight of the sample is 1.2 g. 75 INJECTIO ALGOPYRINI 50% (INJ. ALGOPYRIN 50%) ANALGETICUM. Background: The sodium methylsulfonate group in metamizole sodium salt (noraminophenazone) is hydrolyzed in acidic medium to give 1-phenyl-2,3-dimethyl-4-methylpyrazolone, formaldehyde and SO2. SO2 + H2O H2SO3 H2SO3 HSO3- + H+ HSO3- SO32- + H+ SO32- + I2 + H2O SO42- + 2 I- + H+ Quantitative analysis of metamizol sodium salt: 0.4000 g (about 11-12 drops) of material is weighed into a 50-ml Erlenmeyer flask with a glass stopper. Cool the flask, add a cold mixture of 20 ml of methanol and 3 ml of 50% H2SO4 and then titrate the mixture with standard 0.05 M I2 solution. N.B Keep the temperature below 15 °C (ice-cold water-bath). Do not shake the mixture vigorously as SO2 is volatile. According to the Ph.Eur., under continuous temperature control (ice-cold water-bath) after the addition of 0.01 M HCl metamizole sodium is titrated with 0.05 M I2 solution, with starch as indicator, until the solution turns blue and the color persists for 1 min. 1 ml of standard 0.05 M I2 solution is equivalent to 16.67 mg of water-free noraminophenazone sodium mezylate/metamizole sodium. The density of the injection at 20 ºC is 1.1778 g/ml. Calculate the percentage of metamizole in the sample in g / 100 ml with twodecimal precision. Notes: The quantitative assay is carried out by the addition of 1-2 drops of alcoholic methylene blue and the clear blue solution is then titrated with I2 until it turns emerald-green. 76 ACIDBASE TITRATIONS Acidbase titrations are important in pharmaceutical analysis. Pharmaceutical compounds frequently contain weak acidic or weak basic groups and their analyses are usually unique. The titrations are not always perfomed in aqueous solutions, but often in aqueous alcoholic or alcoholic medium. Very weak bases are analyzed with a mixture of glacial acetic acid and HClO4 in nonaqueueos medium. An acid–base titration curve is a graph of pH against the volume of titrant added during the titration. The shape of the curve is specific for the analysis. The end-point of the titration is the neutralization point, the inflexion point (the steepest slope) in the curve. Dyes are usually used to indicate the equivalence point of acid–base titrations. An acid– base indicator is a compound whose protonated and deprotonated forms have different colors. The pH change is indicated by the color change of the indicator. A small error is introduced because the indicator also consumes titrant, and it should therefore be used in only a small quantity. Moreover the color change can be detected more easily when a small quantity of indicator is used. The choice of the indicator depends on the equivalence point of the titration. The pH range in which the color of the chosen indicator changes should be really close to that at the end-point of the titration. The indicator exists in both dissociated and nondissociated forms at the equivalence point of the titration, and therefore a transition color is visible. Visual detection of the transition color is difficult, because it depends on a number of factors. A color change is noticed by the human eye if one form becomes dominant, which means at least a 10fold excess. A transition interval is therefore indicated, not a transition point. Mixed indicators are sometimes used to make the color change easier to detect. In most of these cases, the original color turns to its complementary color. In one ideal case, red turns to green, when the transition color is gray. The complementary color change can be achieved through the addition of dyes such as methylene blue instead of a second indicator. The acid–base feature of the indicators also depends on the media and the temperature. To decrease the possibility of errors during the analyses, the recommended indicator should be used at very close to room temperature. The features of the indicators are influenced by various factors. The transition of an acidic indicator is shifted in the direction of acidic pH when the temperature increases, while the transition of a basic indicator is shifted in the direction of basic pH. Organic solvents such as alcohols shift the transition of acidic indicators to a more basic pH and of basic indicators to a more acidic pH. Colloid solutions adsorb the indicator and change the color transition. The behavior of indicators is influenced by the presence of CO2 and NH3 possibly dissolved from the atmosphere. Boiling the sample before the equivalence point may be a suitable way to remove CO2 in the case of basic indicators such as phenolphthalein or thymolphthalein, or to remove NH3 in the case of acidic indicators. The sample should then be cooled back to room temperature before the titration is completed. Standard glacial acetic acid–HClO4 solution is used for the analysis of very weak bases, and the titration is performed in glacial acidic acid medium. Such weak bases include lidocaine, benzocaine, procaine, xanthine derivatives such as theophylline, theobromine, etc., and certain sulfonamides that contain primary or secondary amino groups. 77 INDICATOR TRANSITION INTERVAL pKI COLOR ACID BASE Methyl violet 0.1-3.2 yellow violet Cresol red 0.2-1.8 red yellow Thymol blue 1.2-2.8 red yellow Dimethyl yellow 2.4-4.0 3.45 red yellow Bromophenol blue 3.0-4.6 4.06 yellow violet Methyl orange 3.2-4.4 3.76 red orange Bromcresol green 3.9-5.4 4.90 yellow blue Methyl red 4.2-6.2 4.96 red yellow Bromothymol blue 6.0-7.6 7.3 yellow blue Neutral red 6.8-8.0 7.4 red yellow Phenol red 6.8-8.2 7.72 yellow red Cresol red 7.2-8.8 8.08 yellow red Thymol blue 8.0-9.2 8.82 yellow blue Phenolphthalein 8.0-10.0 9.5 colorless red Thymolphthalein 9.4-10.6 9.7 colorless blue Tropaeolin 00 11.0-13.0 yellow orange MIXED INDICATORS PROPORTION TRANSITION PH COLOR ACID BASE Methyl redBromocresol green 1:3 5.1 orange green Methyl red – Methylene blue 1:1 5.3 violet green Phenolphthalein Methylene green 1:2 8.8 green violet Standard glacial acetic acid–HClO4 solution is prepared from a mixture of the most concentrated (70%) HClO4 solution and glacial acetic acid. Acetic anhydride is added to the mixture in an amount equivalent to the water in the solution. As an example standard 0.1 M glacial acetic acid– HClO4 solution is prepared from 8.5 ml of HClO4, 900 ml of glacial acetic acid and 30 ml of acetic anhydride, and the mixture is diluted to 1000 ml with glacial acetic acid. The water content of the standard solution is checked 24 h later (e.g. the Fischer method) and set to between 0.1% and 0.2% with water or acetic anhydride and the mixture is left to stand for another 24 h. The standard solution must not contain acetic anhydride, which would 78 react with primary or secondary amines. The solution is standardized with potassium hydrogenphthalate in the presence of crystal violet as indicator. The temperature of the solution is important during the standardization because of differences in thermal expansion. When an analysys is carried out at a temperature that is different from the temperature of standardization, the volume should be corrected by using the following formula: VCorr=V[1+(T1-T2)·0.0011], where V is the volume measured in analytical titration at T2, while T1 is the standardization temperature. Crystal violet (yellow to violet), cresol green (yellow to red) and methyl violet can be used as indicators with standard glacial acetic acid–HClO4 solution. Tetrabutylammonium hydroxide is used as nonaqueous basic standard solution for the titration of acids. Its standard 1 M solution is commercially available. Its toluene solution is prepared from tetrabutylammonium iodide with Ag2O in the presence of methanol, and then diluted with toluene. Benzoic acid is used in the presence of thymol blue as indicator for standardization directly before use. This is a very sensitive standard solution. It should be kept away from light and moisture. It can be used for potentiometric analysis acids of different strengths, inorganic acids, carboxylic acids and phenols in the same sample. 79 SPIRITUS IODOSALICYLATUS (SPIR. IODOSALIC.) ANTIMYCOTICUM. ANTISEPTICUM Composition: Potassii iodidum 0.10 g Acidum salicylicum 0.90 g Solutio iodi alcoholica 6.00 g Ethanolum 70% 23.00 g Total mass: 30.00 g Ingredients of Solutio iodi alcoholica (Ph. Hg. VII.) Composition: Potassium iodatum 4.0 g 5.0 g Aqua destillata 10.0 g Spiritus contentratissimus 81.0 g 100.0 g Iodum Total mass: Determination of iodine content: Weigh 4.0000 g (4.5 ml) of substance into an Erlenmeyer flask with a glass stopper. Add 10 ml of cold freshly distilled water and titrate the solution with 0.1 M Na2S2O3 solution in the presence of 2 drops of starch solution. Spiritus iodosalicylatus is volatile at room temperature. First tare the Erlenmeyer flask (together with the glass stopper), remove it from the scale, add the 4.5 ml of substance to the flask, and replace the glass stopper immediately. Measure it as quickly as possible. Avoid putting any substance on the side-walls of the flask. The analysis can be performed without the addition of starch, simply by monitoring the disappearance of the color of I2. It is recommended to add starch only at the very end of the titration, when the color of the solution is pale-yellow, and then continue the titration until the blue color disappears. 1 ml of standard 0.1 M Na2S2O3 is equivalent to 12.69 mg of I2. Calculate the mass of I2 in 30 g of substance and give the result in mg with two˗decimal precision. Determination of salicylic acid: Add 3 drops of phenolphthalein indicator to the solution used for I2 analysis and titrate with it 0.1 M NaOH solution. The addition of freshly distilled water is necessary so that this acidbase titration can be carried out properly. During the titration, precipitate of salicylic acid may be observed, which dissolves on the addition of NaOH. Care must be taken to ensure that the, salicylic acid precipitate (needle crystals) should be dissolved completely and not left on 80 the walls of the flask. At the equivalence point, the pink color of the solution should persist for at least 1 min. 1 ml of standard 0.1 M NaOH solution is equivalent to 13.81 mg of salicylic acid. Calculate the mass of salicylic acid in 30 g of substance and give the result in mg with twodecimal precision. Determination of potassium iodide content: This analysis is not included in this semester requirements. Add 5 ml of 1 M H2SO4 and 50 ml of water to the solution used for the analysis of salicylic acid. The mixture is titrated with 0.1 M AgNO3 solution in the presence of metanil yellow. NaO3S N H N N Metanil yellow (red at pH 1.5, yellow at pH 2.7) The AgNO3 is added dropwise at close to the equivalence point until the grayish-blue color of the solution turns cyclamen red and the dye adsorbed on the surface of the precipitate is blue. Subtract the volume measured in the I2 analysis from the volume measured in the KI analysis. 1.00 ml of standard 0.1 M AgNO3 is equivalent to 16.600 mg of KI. This titration is performed with the addition of phenol red (yellowred transition) according to Ph.Eur. 81 TEST YOURSELF – SAMPLE TEST QUESTIONS 1. Which of these phenomena is the most important in UV spectroscopy? a. b. c. d. e. Excitation of the rotation of the molecules. Excitation of the rotation of the substituents. Excitation of the electron system of the molecules. Excitation of the outer electrons of light atoms. Excitation of the inner electrons of atoms. 2. Absorbance is defined by the intensities of the incoming light (I0) and the transmitted light. Which of the following equations is correct? a. b. c. d. e. A= I0/I A=log I0/I A= (I0-I)/I A= (I-I0)/I A=(log I0)/I 3. Which wavelength region is official for IR analysis in the Pharmacopoeia? a. b. c. d. e. 2.5-15 cm-1 60-208 cm-1 206-560 cm-1 670-4000 cm-1 3800-6000 cm-1 4. Which light source is used in atomic absorption spectroscopy? a. b. c. d. e. A tungsten lamp A deuterium lamp A helium-neon laser A Nernst lamp A hollow cathode lamp 5. Which data are necessary to calculate the content of an active substance in spectrophotometric analysis? a. b. c. d. e. The absorbance, the relative absorption coefficient and the length of the cell. The absorbance and the length of the cell. The absorbance and the wavelength of the applied electromagnetic wave. The absorbance and the molecular weight of the active ingredient. The absorbance, the molar absorption coefficient and the length of the cell. 6. What is contained in the calomel electrode? a. b. c. d. e. Hg/HgCl2 An appropriate pH buffer Hg/Hg2Cl2/KCl NaCl solution Ag/AgCl 82 7. Which of the following are found as two units in a double-beam spectrophotometer? a. b. c. d. light source, cell, detector cell cell, monochromator, detector light source, monochromator, detector 8. What happens to the absorption of a compound at the isobestic point? 1. 2. 3. 4. The absorption ceases. The absorption increases. The absorption is unaltered. The absorption decreases. a. b. c. d. e. Only the 1 is correct. Only the 2 is correct. Only the 3 is correct. Only the 4 is correct. None of them are correct. 9. Which data are necessary to calculate the relative absorption coefficient of a substance? 1. 2. 3. 4. 5. The slit width (cm) The path length (cm) a. b. c. d. e. The concentration of the solution (g/100 ml) The molecular weight of the substance. The absorbance of the sample. Only points 1 and 2 are correct. Only points 1 and 4 are correct. Only points 1 and 3 are correct. Only points 1, 3 and 4 are correct. Only points 2, 3 and 4 are correct. 10. Which of these statements are correct? 1. 2. 3. 4. a. b. c. d. The API of Algopyrin injection is determined at room temperature. Standard I2 solution is used, which reduces the API. The sample is shaken slowly to avoid the volatilization of SO2. The color of the starch indicator disappeares at the end-point of the titration. Only point 3 is correct. Only points 1 and 4 are correct. Only points 1, and 4 are correct. Only points 2 and 3 are correct. 11. Which statements are incorrect? 1. Spiritus jodosalicylatus is measured quickly into a titration flask to avoid the evaporation of the alcohol. 2. The salicylic acid content of a sample is determined by acidbase titration with standard HCl solution. 3. Only a few drops of indicator are used because the acidbase indicator does not utilize any standard solution. 4. The end-point of the titration is indicated by the appearance of the pink color of phenolphthalein. a. b. c. d. e. Only points 1 and 2 are incorrect. Only points 1 and 4 are incorrect. Only points 2 and 3 are incorrect. Only points 1, 3 and 4 are incorrect. Only points 2, 3 and 4 are incorrect. 83 12. Which statements are incorrect? 1. The aminophenazone content of Suppositorium antipyreticum is determined by cerimetry. 2. Standard Ce2(SO4)3 solution iss used. 3. The aminophenazone ring was opened by nascent oxygen. 4. Indicator is ferroin. a. b. c. d. e. Only point 2 is incorrect. Only points 1 and 3 are incorrect. Only points 1, 2 and 3 are incorrect. Only points 1 and 4 are incorrect. None of them are correct. 13. 20% of a monochromatic light beam is passed through a solution. What percentage of the incident light I0 is transmitted? a. b. c. d. e. 5% 10% 20% 40% The given data are not sufficient to determine this. 14. Which one of the following are true? The light absorption of a two-component system is described by (the absorbance of the components do not affect each other): a. b. c. d. AAB=AA+AB AAB=AA-AB AAB=log(AA/AB) TAB=TA+TB 15. Which gas can be used as cooling gas in the case of ICP? a. b. c. d. N2 Ar O2 Ne 16. The Na+ content of an infusion can be determined 1. 2. 3. 4. 5. a. b. c. d. e. on the basis of the absorption of Na+ in VIS (yellow light). by flame photometry. by atomic absorption spectroscopy. on the basis of the yellowish-green absorption of Cl- ions. by complexometric titration with EDTA. Only point 1 one is correct. Only points 2 and 3 are correct. Only points 1, 2 and 3 are correct. Only points 2, 3 and 4 are correct. All of them are correct. 84 17. Which statements are true for the complexometric determination of the cation content of Pulvis neutracidus? 1. Side-by-side determination of Bi3+ and Mg2+ is not possible. 2. The sample should be ignited before the analysis because of its glucofrangulin content, which can form complexes with inorganic cations. 3. Bi3+ can be titrated directly after acidic digestion. 4. Eriochrome black T is used in 1% trituration because the indicator utilizes the standard solution. a. b. c. d. e. Only point 2 is true. Only points 1 and 3 are true. Only points 2 and 3 are true. Only points 2, 3 and 4 are true. All of the answers are true. 18. Which statements are true as concerns separation techniques? 1. Al2O3, SiO2 or paper can be used as solid-phase absorbent. 2. A reverse-phase column can be obtained by the transformation of SiO2 to silica chloride. 3. Polar substances pass fastest through a reverse-phase column. 4. A chemical reaction is observed between the analyte and the eluent. 5. Liquids can be used as stationary phase in gas chromatography. a. b. c. d. e. Only points 1, 2 and 3 are true. Only points 2, 3 and 4 are true. Only points 1 and 4 are true. Only points 1 and 5 are true. Only points 2, 3 and 5 are true. 19. The following units correspond to ppm: 1. 2. 3. 4. 5. μg/ml mg/ml μg/dm3 mg/l pg/ml a. b. c. d. e. Only points 2, 3 and 5 are true. Only points 5 is true. Only points 3 and 5 are true. Only points 3, 4 and 5 are true. Only points 1 and 5 are true. a. b. c. d. e. Only points 3 and 4 are true. Only point 2 is true. Only point 3 is true. Only points 2, 3 and 4 are true. Only points 1, 2 and 5 are true. 20. The following units correspond to ppb: 1. 2. 3. 4. 5. μg/l pg/l ng/dm3 pg/cm3 μg/cm3 85 21. Which statements are true for atomic absorption spectroscopy? 1. 2. 3. 4. 5. All the elements can be analyzed by this technique in ppm concentration. Some non-metallic elements can be analyzed by this technique. It may help the diagnosis of Wilson’s syndrome. The sample is solvated during the atomization. 0.001% relative precision can be achieved. a. b. c. d. e. Only point 1 is true. Only points 2 and 4 are true. Only points 2 and 3 are true. Only points 1 and 4 are true. All of the answers are true. 22. Oxidation is experienced when an acetylene-air flame is used for atomic absorption. How can the problem be solved? 1. The proportion of acetylene should be decreased. 2. 3. The proportion of air should be decreased. 4. 5. The proportion of fuel gas should be increased. The proportion of combustive gas should be increased. The proportion of combustive gas should be decreased. a. b. c. d. e. Only points 1, 3 and 5 are true. Only points 2, 3 and 4 are true. Only points 1, 3 and 5 are true. Only points 2, 3 and 5 are true. Only points 1, 3 and 4 are true. 23. Which statements are true for the determination of protein concentration? 1. Both UV and VIS spectroscopy can be used. 2. The applied dyes shift the spectrum to lower wavelengths. 3. The oxidation of copper ions is utilized in several techniques. 4. Fluorimetry can be applied. a. b. c. d. e. Only points 1, 2 and 4 are true. Only points 1, 2 and 3 are true. Only points 2 and 3 are true. Only points 2 and 4 are true. Only points 2, 3 and 4 are true. 24. Which statements are true for the classical titration of Suppositorium antipyreticum? 5. Standard Ce(SO4)2 solution is used that is diluted in H2SO4 to maintain its stability. a. Only points 1, 2 and 4 are true. b. Only points 1, 2 and 3 are true. c. Only points 2, 4 and 5 are true. d. Only points 2 and 4 are true. e. Only points 2, 3 and 5 are true. 1. It is a reductometric titration. 2. The cerium ion undergoes a change in its oxidation number. 3. The oxidation number of cerium increases. 4. The oxidation number of cerium decreases. 86 25. Which statements are not true for atomic absorption spectroscopy? 1. 2. 3. 4. 5. Tungsten and hollow cathode lamps can be used as light source. Contamination originating from the die during tableting can be detected. Any element can be measured in solution. ICP can be used for the excitation of the atoms. Individual light sources are necessary to the measurement of each element. a. b. c. d. e. All of the answers are false. Only points 1, 2, 3 and 4 are false. Only point 5 is false. Only points 2, 3 and 5 are false. Only points 3 and 4 are false. 26. Which statements are true? 1. 2. 3. 4. 5. pH is directly proportional to the emf. The absorbance depends on the length of the light path. pH is inversely proportional to the emf. The conductivity is independent of the surface of the electrode. In determinations of the molar relative conductance, the conductances of the individual ions are additive. a. b. c. d. e. Only points 1, 2 and 5 are true. Only points 2, 3 and 4 are true. Only points 1, 4 and 5 are true. Only points 2, 3 and 5 are true. Only points 1, 2 and 4 are true. 27. Which statements are false? 1. 2. 3. 4. 2 ppm is the same as 2 mg/l. 4 ppb is the same as 4 μg/l. 2.5 μm is the same as 400 cm-1. 1.0 absorbance is the same as 90% transmittance. 5. 5 ppm is the same as 5 μg/l. a. b. c. d. e. Only points 2 and 3 are false. Only points 1 and 4 are false. Only points 3 and 4 are false. Only points 2 and 4 are false. Only points 4 and 5 are false. 28. According to the Pharmacopoeia Hung. VIII, which compounds are analyzed by direct conductometry? a. b. c. d. e. Alkaline compounds. Distilled water. Benzoic acid. Acetylsalicylic acid. Acidic compounds. 29. Which effects shift the electromagnetic radiation absorption maximum of a compound to higher-energy wavelengths? a. b. c. d. A bathochromic shift. A hypochromic shift. A hypsochromic shift. A hyperchromic shift. 87 30. What is the mathematical relationship between the conductance of a solution and the distance between the electrodes? a. The conductance of the solution is directly proportional to the distance between the electrodes. b. The conductance of the solution is inversely proportional to the distance between the electrodes. c. The conductance of the solution is directly proportional to the square of the distance between the electrodes. d. The conductance of the solution is directly proportional to the logarithm the distance between the electrodes. 31. An alkaline solution is titrated with a standard acidic solution. Which of the following statements are true for the titration curves? a. A local minimum is observed at the equivalence point in the case of the first derivative. b. A local minimum is observed at the equivalence point in the case of the second derivative. c. The second derivative is zero at the equivalence point. d. A local maximum is observed at the equivalence point in the case of the second derivative. e. A local maximum is observed at the equivalence point in the case of the first derivative. 32. 0.10 g of substance is dissolved in 100 ml of methanol. 5 ml of 0.1 M Na2CO3 is added to 0.5 ml of the solution, which is diluted 100 ml. What blank solution should be prepared? a. b. c. d. e. Methanol is the blank solution. 0.1 M Na2CO3. 0.01 M Na2CO3. 0.02 M Na2CO3. 0.005 M Na2CO3. 33. Phenolphthalein is diluted with Na2CO3 solution. What effect can be observed? a. b. c. d. A bathochromic shift. A hypochromic shift. A hypsochromic shift. A hyperchromic shift. 34. How does the number of ions change after the equivalence point in the conductometric titration of a weak acid and a weak base? a. It increases. b. It does not alter. c. It decreases. 88 35. How does the number of the ions change before the equivalence point conductometric titration of a strong acid and a strong base? a. It increases. b. It does not alter. c. It decreases. 36. What kind of chemical reaction takes place between the peptide bonds in proteins and the biuret reagent? a. b. c. d. e. Oxidation. Reduction. Complex formation. Substitution. Addition. 37. What is the first step in a photometric measurement? a. b. c. d. Determination of the absorption of the sample. Determination of the absorption of the calibrating solutions. Determination of the absorption maximum of the sample. Determination of the baseline. 38. How does the slope of a calibration curve change when less is measured of the reference material compared with the given value? a. The slope of the calibration curve increases. b. The slope of the calibration curve does not change. c. The slope of the calibration curve decreases. 39. What kind of light source can be applied in the wavelength region 380-780 nm? a. b. c. d. A deuterium lamp. A ceramic rod. A tungsten lamp. A halogen lamp. 40. Which statements are false for the titration of Unguentum ad vulnera? a. Salicylic acid is titrated directly with standard NaOH solution. b. The volume relating to H3BO3 is calculated by subtraction oftthe values at the first inflexion point from the value at the second inflexion point. c. The complexes of H3BO3 with polyols decrease the pH. d. H3BO3 is titrated as a trivalent acid. 41. The conductance of Aqua purificata is: a. b. c. d. 0; it should not contain any contamination. 1.1 μS/cm. 3.4 μS/cm. 4.3 μS/cm. 89 42. Which statements are true? 1. Standard I2 solution is used for the titration of oxidative substances. 2. Standard I2 solution can be used for the titration of the antioxidant ascorbic acid. 3. During the titration of metamizole sodium, the resulting formaldehyde is oxidized to formic acid by I2. 4. The end-point of Algopyrin inj. titration is indicated by the appearance of the blue color of starch. 5. The sample is cooled during the titration of Algopyrin inj. because the resulting H2SO4 should not be warmed up. a. b. c. d. e. Only points 1, 2 and 3 are true. Only points 1, 2, 3 and 4 are true. Only points 3 and 5 are true. Only points 2 and 4 are true. All the answers are true. 43. Which statements are true for the retention time? 1. 2. 3. 4. The retention time is the time required for the entire separation of a sample. The retention time is different for all substances. The retention time is the most important parameter of a chromatogram. The retention time is the time between the application of the sample and its appearance in its maximal concentration. 5. The retention time is also called the back-titration time. a. b. c. d. e. Only point 1 is true. Only points 1, 2 and 3 are true. All the answers are true. Only points 2, 3 and 4 are true. Only points 2, 3 and 5 are true. 44. Which statements are not true for atomic absorption spectroscopy? 1. 2. 3. 4. 5. Tungsten and hollow cathode lamps can be used as light source. Contamination originating from the die during tableting can be detected. Any element can be measured in solution. ICP can be used for the excitation of the atoms. Individual light sources are necessary for the measurement of each individual element. a. b. c. d. e. All of them are false. Only points 1, 2, 3 and 4 are false. Only point 5 is false. Only points 2, 3 and 5 are false. Only points 3 and 4 are false. 45. How can an exact standard NaOH solution be prepared? a. An exact NaOH standard solution cannot be prepared. It should be standardized before use. b. NaOH should be measured directly. c. Through the direct measurement of NaOH in an inert gas atmosphere. d. Through the direct measurement of NaOH at 0% humidity. 90 46. Which statements are true for the determination of protein concentration? 1. Both UV and VIS spectroscopy can be used. 2. The applied dyes shift the absorption maximum of the electromagnetic radiation toward lower-energy wavelengths. 3. The oxidation of copper ions is utilized in several techniques. 4. Biuret or BCA reactions are good examples of protein concentration determination. a. b. c. d. e. Only points 1, 2 and 4 are true. Only points 1, 2 and 3 are true. Only points 2 and 3 are true. Only points 2 and 4 are true. Only points 2, 3 and 4 are true. 47. Which statements are not true for atomic absorption spectroscopy? a. b. c. d. Tungsten and hollow cathode lamps can be used as light source. The Pb contamination of the medicinal plants can be determined by this method. Any element can be measured in solution. Individual light sources are necessary for the measurement of each individual element. 48. Which statemens are not true? a. Steroids can be quantitated by UV-VIS photometry after their condensation with amines. b. Picrate salts absorb UV light. c. Acetylsalicylic acid absorbs UV light. d. A quartz cell is used for measurements in the UV range. 49. What can disturb photometric measurements? a. b. c. d. e. Fingerprints on the wall of the cell. Suspensions. Solvent on the wall of the cell. Bubbles in the sample solution. All of the above. 50. What are the advantages of photometric measurements? a. b. c. d. e. Indicators can be excluded. Dark solutions can be titrated. Precipitation titration is possible. They are more accurate than traditional classical titration. All of the above. 91 APPENDIX UNICAM UV/VIS SPECTROPHOTOMETER MANUAL 1. Turn the spectrophotometer on 15 min before the measurement. (The ON/OFF button is on the left-hand side of the instrument.) 2. Recording a spectrum, and determination of the absorption maximum: a. Scan, then ENTER. b. Check the following parameters: i. Mode: It should be ABS. ii. Start: EDIT. Give the shorter wavelength of the scanning range, then ENTER. iii. Stop: EDIT. Give the longer wavelength of the scanning range, then ENTER. iv. Peak table: EDIT→PEAKS→ENTER. 3. Choose the appropriate measuring cell: a. UV range: quartz cell. b. VIS range: glass or plastic cell. 4. Determination of the absorption maximum: a. Place the cell containing the reference solution into both slots of the spectrophotometer. The light comes from the back to the front. b. Push ZeroBase and record the baseline throughout the chosen spectral range. c. Put the cell containing the third calibration solution into the sample slot. Keep the reference solution in the reference slot. d. Start the measurement with RUN. e. View results: 5th gray button under the screen. Record the value of the absorption maximum. 5. Go back to the main menu: HOME. 6. Choose Quant, then ENTER. 7. Determination of the calibration curve: a. Set the correct wavelength: i. Wavelength→EDIT→give the value of the absorption maximum, then→ENTER. b. Set the parameters of the calibration: i. No Standards, number of calibration solutions: EDIT→5→ENTER. ii. Standards (first gray button under the screen), then EDIT, give the concentrations of the calibration solutions, then ENTER. iii. Calibrate (third gray button under the screen), then ENTER. iv. Place the first calibration solution into the sample cell, then RUN. v. Repeat the measurement with all the other calibration solutions as in point iv. 93 vi. View results: Check the equation of the calibration curve, and note the value of the coefficient. 8. Determination of the sample concentration: a. Place the sample into the sample cell, RUN. 94 UV-1601 SHIMADZU SPECTROPHOTOMETER MANUAL 1. Turn the spectrophotometer on 15 min before the measurement. (The ON/OFF button is on the left-hand side of the instrument.) 2. Recording of the spectrum, and determination of the absorption maximum: a. Choose Spectrum, 2 from the numerical characters. b. Check the following parameters: i. 1. Measurement mode: ABS. ii. 2. Scanning range: give the appropriate wavelength range, depending on your sample: 1. 2 from the numerical characters 2. Give the longer wavelength of the scanning range, then ENTER. 3. Give the shorter wavelength of the scanning range, then ENTER. 3. Choose the appropriate measuring cell: a. UV range: quartz cell. b. VIS range: glass or plastic cell. 4. Determination of the absorption maximum: a. Place the cells containing the reference solution into both slots of the spectrophotometer. The light comes from the left to the right. b. Push the F1 button BaseCorr: for baseline correction throughout the chosen wavelength range. c. Place the cell containing the middle calibration solution into the closer sample slot. d. Start the measurement with the Start/Stop button. e. Analyze the data with the DataProc F2 button. f. To obtain the peak(s) at the absorption maximum, push button 3 Peak and note the value(s). 5. Go back to the main menu: a. RETURN b. RETURN c. RETURN d. MODE. e. Current data not saved. f. OK→F3 button. 6. Choose: „Quantitation” button 3. 7. Determination of the calibration curve: a. Set the correct wavelength: i. Button 1 95 ii. Button 1 again iii. Give the correct value, then ENTER. b. Set the parameters of the calibration: i. Button 2 Method ii. Button 3 Multi-point calib. 1. No of Std. Number of standards: 5. 8. Determination of the calibration curve: a. First push the Start/Stop key. b. Give the concentrations of the calibration solutions. Push ENTER after each value. c. Button 2 Meas (only cell 1): place the first solution into the sample slot (the closer one); the cell containing the reference solution is kept in the reference slot, and the measurement is started with the Start/Stop key. Repeat this step with all the calibration solutions. d. Draw the calibration curve with F1 CalCurb. e. Get the equation of the calibration curve with F4 and note the value of r2. f. RETURN (twice). 9. Determination of the sample concentration: a. F3 SamplMeas. b. Start/Stop. 96 MARS CEM MICROWAVE DESTRUCTOR MANUAL 1. Place the sample into the container of the microwave destructor and add the appropriate amount of acid. Strive to wash all the sample off the wall of the container. 2. Keep the blue valve open and place the lid on the container. 3. Close the blue valve, place the containers into the special holders and fix them. 4. Turn the machine on. (The button is at the right-hand side of the machine.) 5. Choose the appropriate program: a. Load Method→Select b. User directory→Select c. GYAK-HP500→Select 6. Place the samples into the microwave destructor. N.B. Never start the program without the reference container. 7. Green button→START The program used by the microwave oven: Time (min) 5 10 15 20 25 30 Power (%) 80 80 100 100 100 100 The table indicates the power as the percentage of the maximum 400 W. The microwave oven is controlled by the pressure measured in the reference valve. The maximum pressure used by the program is 80 psi (5.5 bar). The powertime curve of the microwave oven 97 ATOMIC ABSORPTION SPECTROMETER MANUAL 1. Turn the compressor on AA1 in the preparation laboratory. 2. Check the pressure in the atomic absorption laboratory on AA2 and wait until it reaches 4 bar. 3. Open the acetylene gas valve on the top (yellow: AA3). Check the pressure in the cylinder (the left indicator should not go below 3 bar) and check that the pressure goes to the spectrometer (the right indicator should not go above 1.5 bar). 4. Turn on the extractor hood. 5. Turn on the atomic absorption spectrometer: AA4. 6. Turn on the computer: AA5. 7. Turn on the printer: (AA6). 8. Click on the AA INST.exe icon. Choose the FLAME method. Wait until the background of the 4100 icon at the bottom of the screen turns dark-blue. Choose MANUAL. 9. Choose the experimental element: step 1: Defaults, step 2: choose the correct element and step 3: OK. 10. Click on the Windows menu, choose the Align lamps function and set the correct current intensity for the lamp. (The parameters can be found on the lamps.) 11. Set the energy to the maximum with the white screw on the lamp and accept it by clicking AGC/AIC. 12. Enter the Flame Control menu, and click on the Flame icon to ignite the flame. Check the gas ratio, which should be: C2H2: 2,5; air: 8,0. 13. Enter the Windows menu again, click on Element parameter and then on Instr. And check: a. Set Slit to 0.7 (Height High). b. Set the time of the measument to 5 s. c. Set the BOC to 2 s. d. Set the Read delay to 2 s. e. Repeat the sample and the standards twice. f. The Burner move should be in the ON position. 14. Enter the Element Parameter and choose the Calib menu. a. The first calibration solution is the reference solution. b. Set the standards as st1, etc. c. The second column contains the concentrations of the calibration solutions. Indicate them to at least three decimals. 15. Enter the Windows menu, and choose ID Weight parameter. Give the sample parameters. 16. Choose Flame Control again. Click on Find Reference and put the capillary into the reference solution, then into st2 according to the commands. 98 17. Enter the Windows menu, choose Continous graphics and put the capillary into the reference solution. Push key F3 to set zero absorbance. Put the capillary into st2 and set the absorbance influencing the suction. (Here the 0.3 mg/l standard solution is used and the absorbance in this case should be about 0.182; ±20% difference is acceptable.) 18. Enter the Calibration menu. a. Put the capillary into the reference solution, and click on autozero. Repeat this measurement twice to stabilize the zero value. b. Measure the absorbances of the standard solutions. Wash the capillary with distilled water before each measurement. c. Check the value of the coefficient after the calibration. The closer the value is to 1 is the better the calibration curve. d. Print the calibration curve File menu, then Print Image. 19. Put the capillary into the sample. Start the measurement by pushing F4. The spectrometer measures the samples twice. Wash the capillary with distilled water before each sample. The results are recorded and printed out. 20. Turning off: a. Close the gas cylinder (wait until the indicators show zero) b. Enter the File menu and then Quit to Desktop c. Turn off the spectrometer: AA4 d. Turn off the extractor hood e. Turn off the printer: AA6 f. Enter the File menu and then Exit to DOS g. Turn off the compressor: AA1 99 HPLC MANUAL How to start up the HPLC The in-line filters should be dried with a paper tissue and then placed into the appropriate solution: Solvent line A: 50 mM phosphate buffer pH 6.3. Solvent line B: acetonitrile. Turning on the (SHIMADZU Prominence UHPLC system) instrument: 1. the pump (LC-20AD) (N.B. the degasser is turned on together with the pump and initially gives an error signal); 2. the thermostat (CTO-20A); 3. the detector (SPD-M20A); wait 2 min to allow the vacuum pump of the degasser (DGU-20ASR) to ensure the optimal vacuum. Preparation of the analysis program with the LC Solution software Double click on the blue icon of LC Solution. The LC Solution Launcher starts. Click on the Operation option in the blue window, where the Analysis Series HPLC 1 icon should be selected. A pop-up window appears. User ID: Admin, Password: leave empty, and then OK. Two whistles are heard and a gray window will show: Connecting the LC Instrument… An application appears in the Data Acquisition window: LC Real Time Analysis. Wait until the LC, PDA connection is switched to Ready (green background). Now prepare the project folder: Choose the File menu, Select Project (Folder)...; the Project (Folder) Selection window appears, where the Panadol próba folder should be selected. The program marks the folder in gray color. Click on New Folder: the Create New Folder window appears; write the name of the folder, e.g. “Moday morning 2015” into the line of Please input new folder name. Choose the new folder with a single click in the Project (Folder) Selection window (gray background). Close the dialog window. Creating method file: 1. File menu, Open Method File. 2. A dialog window pops up. Location: Choose the Panadol próba folder and then the Hallgató folder. Double click on the mérés gradiens method file. Save the opened method under a new name. 3. File menu, Save Method File As...: type the name of the method file (e.g. practice-Monday), then Save. Description of the HPLC method. The conditions of the analysis are to be seen in the Instrument Parameters View window. N.B. Here these parameters can be modified, but not now! Click on the Normal button and the Simple Settings option shows the duration of the analysis (Time Program, LC Stop Time: 5.00 min). The PDA is active; the end of the data acquisition is also 5 min (End Time: 5.00 min). Information on the pumps is included here. Pumps: Mode: Low pressure gradient. This means that the analysis is performed in gradient mode; the instrument mixes the solvents according to the program, which changes linearly in time. At present the Total Pump A Flow: 0.000 ml/min, and concentration of Solvent B: 10.0%. The gradient program is as follows: 100 Time Module Action Value 1. 0.01 Pumps Concentration of B 10 2. 4.00 Pumps Concentration of B 20 3. 4.10 Pumps Concentration of B 10 4. 5.01 Stop This means that the initial 10% acetonitrile concentration increases to 20% by the 4th min and then changes back to 10% in 0.1 min. By using a gradient and gradually increasing the proportion of the organic component, a shorter retention time can be achieved. Further, the wide peaks resulting in isocratic elution can be reduced by using gradient elution. By clicking on the Normal mode LC Time Prog. option, information becomes available about the PDA (PhotoDiode Array). Lamp: D2 & W means that deuterium and tungsten lamps are used. Wavelength: Start wavelength: 268 nm; End wavelength: 272 nm. 270 nm is used for data acquisition, but it is worth having a wavelength interval. Full spectra (190-800 nm) should not be measured, because extremely large data files with very much unnecessary information would be created. The optimal wavelength is determined in advance: 270 nm. Paracetamol has an absorption maximum at 238 nm, but the measurement is performed at 270 nm, i.e. the absorption maximum of caffeine. The spectrum of Paracetamol in the interval 200-400 nm The spectrum of caffeine in the interval 200-400 nm The sample contains less caffeine than paracetamol (approximately one-eighth of its amount). The diluted sample contains trace only amounts of caffeine, and therefore you have to set the wavelength to the absorption maximum of caffeine to determine the concentration of 101 the active ingredients correctly. Paracetamol absorbs UV light at 270 nm with sufficient intensity (~700 mAU), and this wavelength is therefore optimal for the detection during the HPLC analysis. Preparation of the HPLC system for the measurement: 1. Purge: removal of bubbles from the system with high flow speed. The valve to the column is closed. The purge valve in the pump unit should be opened by turning the knob in the anticlockwise direction twice (the first turn is difficult). The value of Solvent B Conc. should be changed from 10% to 50% in the Normal mode Simple Setting option to degas both of the solvent lines. The value of Total Pump A Flow should be altered to 5 ml/min. (N.B. Only when the purge valve is open!) Click on DownLoad. A dialog window pops up: Current Method File will be Saved. OK. The Oven ON/OFF and the Pump ON/OFF should now be ON with a single click on the appropriate buttons on the instrument control panel. Leave the system in operation mode for 2 min after the high flow has started. Two minutes later, the flow rate should be zero again: Total Pump A Flow: 0 ml/min, DownLoad, OK. Close the purge valve. The Concentration of Solvent B should be 10% again (DownLoad, OK). 2. Increasing the flow rate gradually One of the goals is to wash the column with the solvent and the other is to reach the flow rate used during the measurement. It is advisable to alter the flow rate gradually to protect the column. The pressure increases when the flow rate is increased, and the stationary phase might therefore be damaged if the highest flow rate is used immediately. Small increments are applied. The flow rate should be increased in 0.2-ml/min steps until the final 1.5 ml/min is reached. All steps should be held for 2 min to stabilize the pressure. The following values are set: 0.2, 0.4, 0.6, 0.8, 1.0, 1.3, 1.5 ml/min. N.B. The program accepts only decimal points. The values of the pressure and the flow rate can be followed in the data acquisition window. The uppermost yellow area shows the changes in pressure and intensity with time. The window can be widened if necessary. Currently it is 5 min wide. Click the right-hand button on the yellow area and choose Display Settings, General option in the pop-up window. The time in the Time Range should be overwritten to 60 min then OK. The steps are now visible. Check the second window if 270 nm is indicated. If not, overwrite the value. Click the right-hand button on the yellow area and choose Display Settings, PDA option. Overwrite the value of the wavelength to 270 nm in the first field (Wavelength), then OK. 3. Washing the column The column should be washed for an additional 30 min after the final 1.5 ml/min flow rate has been reached. This step is necessary to equilibrate the column and to reach a dynamic balance between the stationary and mobile phases. Meanwhile, the Batch file should be prepared. 4. Preparation of the Batch file The measurementmust now be planned. The program works according to the samples; it evaluates them on the basis of the parameters specified here. The options of the analysis were specified earlier (in the Method file). The program applies these in the case of each sample. Click on the Batch Processing icon on the left side of the screen (additional tool bar). Batch 102 view becomes visible. File menu, New Batch File. The actual batch file can be prepared in the empty table. File menu, Save Batch File As… Write the chosen name (e.g. practice-Monday), OK. Data should only be entered into the following columns: Sample Name, Sample Type, Analysis Type, Method File, Data File, Level, Inj. Volume. Fill in the table according to the example below: 103 Sample Name: Enter the name of the samples. First a blank solution is injected (solvent), and then the standards for the calibration routine. Standard 1 will be injected 5 times. Name the standards, for example s11, s12..., Standard 2 then will be injected 2 times to determine the correlation. Finally enter the name of the samples, e.g. the name of the student. Sample Type: the blank remains unknown 0: Unknown. The next sample is the Standard1 solution, injection 1. This is the first calibration solution. Click on the arrow in the right corner of the area. Indicate the sample as Standard in the pop-up window, then choose the Initialize Calibration Curve among the calibration types, OK. In the Sample type, Standard is indicated and “I” in brackets as Initialize. The next sample is the Standard1 solution, injection 2. Scroll down the arrow. This sample is also a Standard, but it is not initial, so click on Add Calibration Level icon and then OK. The sample has now been added to the calibration series, but not as an initial one. It is indicated as a Standard only. This step is repeated with Standard1 to reach sample 5. The calibration solutions of Standard2 and the type of the samples remain unknown, 0: Unknown. Analysis Type: everything remains IT QT (IT: Quantitative Integration, QT: Quantitative Calculation). Method file: the name of the previously saved file appears in the first line if everything was properly done. All the other lines should be filled by scrolling down the arrow and choosing the appropriate method file. N.B. It is not possible to copy/paste the options. Be careful to select the appropriate method file and check that the extension is .lcm. Data File: the lines should be filled in individually. Click on the right-hand arrow and the Select Data File window opens. Write the name of the file.Iit is advised to use the same name as the name of the sample. Save. “.lcd” extension is added to the data file. Be careful: if the line is filled, the data file will not be created with “lcd” extension and it cannot be saved. Level#: remains 1. All lines should be filled in individually. Inj. Volume: 20 should be indicated here as the injected volume from the loop onto the column. The loop used by the instrument is 20 µl. This is the volume that reaches the column from each sample. The optimal circumstances of the quantitative determination are now defined. Save the batch file when all the boxes are filled. Click on the Floppy/Save icon on the toolbar. Click on the Data Acquisition option below again. Check the washing program. Prepare for the sample injections. It is important to inject the samples one after an other. When one sample is completed (5 min), do not hesitate, but inject the next one. It is important to inject the samples rapidly with the same time intervals in the case of gradient elution. It is advisable to prepare the next sample while the previous one is running. The sample loop cannot be filled with the sample while the sample is running. 5. Injection of the sample The instrument is supplemented with a Rheodyne manual injector. The samples are injected manually with a Hamilton pipette. The Hamilton #825 microliter pipette is used during the practicals. The pipette can hold 250 µl, and the tip is replaceable. The tip is a blunt-end tip. The pipette is designed to guarantee a firm grip. The pipette should be rinsed before each injection. You may begin with the blank solution. Approximately 250 µl of solution should be sucked by any pipette slowly so as to avoid bubble formation. The tip of the pipette should be elevated and the bubbles should be removed before injection. It is advisable to use a paper tissue to prevent the solution spraying out. The bubble-free solution (approx. 200 µl) should be emptied into the waste bottle. After the pipette has been washed, another aliquot is sucked up and bubbles are removed if necessary. The volume of the solution should be at least 200 µl. The loop too should be washed. The volume of the loop is 20 µl, so the 200 µl of solution in the pipette is enough for the washing. The tip of the pipette should be placed into the injection valve and pushed until it stops. The injector valve is in the load position. The sample is injected slowly into the loop. If the process is correct, no solution appears at the tip, but the excess amount overflows to the waste. If solution appears at the tip, the whole process should be repeated. After a 30-min washing at 1.5 ml/min, the analysis can be started. 6. Analysis The first sample (the blank solution) is now in the loop. Switch back to the Batch table in the software. Click on the Batch start in the supplementary tool-bar to start the analysis. The screen now shows the analysis view. In the upper part of the screen, the actual view of the Batch table is visible. The sample that is running at the moment is highlighted in gray; all the others are yellow. Check the uppermost yellow area, which shows the changes in pressure and intensity over time. Narrow the window if necessary to be 5 min wide. Click the right-button on the yellow area and choose Display Settings, General option in the pop-up window. The time should be overwritten in the Time Range to 5 min then OK. The following two steps should be executed without any delay. A pop-up window appears: Data Acquisition Start. Click on the Start button, and then immediately turn the valve into the Inject position with a direct movement. After this, 20 µl of sample is injected into the column. The solvent flows continuously through the column and the separation starts. The middle window shows the chromatogram (green line). When the blank solution is running, no peaks appear. For all of the other samples, two peaks are expected after the void volume: first the paracetamol peak, and later the caffeine peak. The next sample is prepared during the run. The Batch view always shows the next sample. Now wash the pipette with Standard1 solution and then prepare the 200 µl of sample for injection. This preparation allows you to inject the new sample immediately after the previous sample. The Batch table switches to the next line when a sample is completed, and the Data Acquisition window pops-up, asking for the next sample. The injection valve should be turned back to the Load position. Inject the sample. Click on the Start button on the screen. The injection valve is turned to the Inject position again. These steps should be alternated until the last sample has been run. After the analysis is finished and all the samples show two peaks (but not the blank solution), the instrument should be stopped. The flow rate should be changed to 0 ml/min. Wait until the pressure becomes 0. Then turn off the instrument in the following order: detector, thermostat, pump. 7. Evaluation of the data The next step is the analysis of the chromatograms. You need to know the area under the curve to determine the concentration of the active ingredient. The program identifies the peaks on the basis of their retention times. To determine the peak areas, the peaks should be assigned, and the integration parameters should be set. This setup was completed previously. Go back to the LC Solution Launcher. Start the Postrun application. The LC Postrun Analysis view appears. Project in: Select Browse Folder... open the yellow folder and choose the newly created folder; double click in Project(Folder) Selection. The window can now be closed. The data from the measurement are uploaded and the data files become visible. You may begin with the standards. Double click on the Standard1 1 file to open the data. New windows will pop up in several views. The chromatogram will be visible in the Chromatogram view. The calibration curve can be seen below. The values of Compound Table View are used for the analysis. The table contains the data relating to the peaks of 106 Paracetamol Name of the sample Retention time Area Retention time Area S11 S12 S13 S14 S15 S21 S22 Caffeine Name of the sample S11 S12 S13 S14 S15 S21 S22 paracetamol and the caffeine. Click on the Results. Two data are necessary and those should be registered into the table above (Ret. Time, and Area). You should record all the values of the standards and the results of your own samples. Close Postrun view. 8. Determination of the active ingredient content of the sample; checking the calibration in the Excel table Double click on the (Eredmények) Results icon on the desktop. Copy the Minta Sheet to the end of the series and rename the sheet, e.g. English-Monday. This new datasheet is used to evaluate the results. Data must be input into the active ingredient content calculation table. Be careful to enter data only into the light-blue cells. The other cells contain equations and if these are changed, incorrect results will be calculated. 107 The paracetamol peak areas of the S1 injections should be written into cells C7-C11 of the table. The caffeine peak areas should be written into cells C29-C33 of the table. The calculated Standard1 paracetamol concentration should be entered into cell D7, and the caffeine concentration into cell D29. The paracetamol peak areas of the Standard2 injections should be written into C20 and C21, and the caffeine Standard2 areas into G28 and G29. The calculated concentrations of these injections should be written into cells D20 and H28. Write the data only into the cells mentioned above. The input of the data on the samples starts with the peak areas of the chromatograms. The peak areas of the samples of paracetamol and caffeine should be written into the table. The next column contains the Labeled Claim/LC value in mg for the analyzed active ingredient. The weights of the samples should be written into the next column, with four-decimal accuracy. Be careful to give the same weights for both active ingredients as the same sample was used in both analyses. The reference (Average tablet weight in g) value should remain at 0.697, the value was used in the calculations. The following data can be found in the calculated cells: RSD%: the relative percentage of the average peak area deviation of the standard1 injections. If this is below 2.0%, the deviation of the injections is appropriate. If it is higher than 2.0%, discuss the situation with the tutor to find out what the problem is. The parameters of the calibration curve are in cells B13-C15 and B35-C37. To calculate the slope, these values will be used and the intersection with the axis should be zero. Cells C23 and G31 show the correlation value and indicate wherter the calibration is correct. If the absolute value is higher than 2.0 in any of these cells. Discuss the situation with the tutor. The value is zero in the ideal case when the standard2 concentration calculated from the calibration is equal to the theoretical concentration (based on the weight of the sample). Column L contains the concentration of the active ingredient, based on the calibration curve. Column M contains the mass percentage of the active ingredient in one tablet. Column N contains the amount of the active ingredient in the tablet in mg. The individual printed out results should be shown to the tutor. 108 NMR SPECTRA THE NMR SPECTRA WERE PROVIDED BY: DR. HABIL. PÉTER FORGÓ 170 160 150 140 10 13 130 9 120 110 8 100 110 7 90 6 80 5 70 60 4 50 40 20.90180 11 39.73320 39.59150 39.45160 39.31150 39.17440 12 3.03 1.03 1.01 1.02 1.00 0.71 O 126.13880 124.16330 123.85840 13 133.85780 131.49730 150.31440 165.75820 169.30910 3 30 2.2461 2.4996 7.9618 7.9489 7.6308 7.6180 7.6050 7.3756 7.3630 7.3503 7.1952 7.1818 1 H NMR SPECTRUM OF ACETYLSALICYLIC ACID acid acetylsal. dmso - 600 O OH O CH3 ppm C NMR SPECTRUM OF ACETYLSALICYLIC ACID acid acetylsal. dmso 600 ppm 160 150 140 130 N O 6.0 13 120 5.5 110 100 N CH3 5. 0 90 111 4. 5 80 4.0 70 60 3.5 50 40 3.3588 7.4719 7.4590 7.4461 7.3439 7.3312 7.3305 7.2739 7.2615 7.2493 3. 0 30 2.98 2.5 20 2.1671 2.4999 2.6555 N 6.01 CH3 2.9064 CH3 2.98 1.08 2.00 1.98 1.00 H3C 9.98560 6. 5 43.47180 39.73170 39.59070 39.45150 39.31210 39.17360 39.03190 36.36210 125.51310 122.68960 122.20010 7.0 128.82390 7.5 135.25810 150.76490 162.65600 1 H NMR SPECTRUM OF AMINOPHENAZONE aminofenazon dmso - 600 ppm C NMR SPECTRUM OF AMINOPHENAZONE aminofenazon dmso - 600 ppm 1 H NMR SPECTRUM OF BENZOIC ACID 7.9805 7.9794 7.9673 7.9653 7.5708 7.5585 7.5461 7.4644 7.4513 7.4386 2.03 1.00 2.06 benzoesav dmso - 600 O 0.75 OH 13. 0 12. 5 12. 0 11. 5 13 11. 0 10. 5 10. 0 9. 5 9. 0 8.5 8.0 7.5 ppm C NMR SPECTRUM OF BENZOIC ACID 170 165 160 155 150 145 112 140 135 130 128.62750 129.46220 130.97390 132.91050 167.56480 benzoesav dmso 600 ppm 150 140 130 6. 5 13 120 110 6. 0 100 5. 5 90 5. 0 80 113 4. 5 70 4. 0 60 50 40 14.19420 7. 0 39.72610 39.58470 39.44500 39.30670 39.16760 N 4.3644 4.3556 4.3470 5.0507 5.0415 5.0323 7.9949 3.24 2.4999 2.4536 3.4516 3.4482 OH 0.37 NO2 3.7072 3.6985 3.6894 3.6808 N 2.19 2.19 1.09 1.00 H3C 48.29270 7. 5 59.81160 132.91650 8. 0 138.40770 151.96050 1 H NMR SPECTRUM OF METRODINAZOL metrodinazol dmso - 600 3. 5 3. 0 30 ppm C NMR SPECTRUM OF METRODINAZOL metrodinazol dmso 600 20 ppm 160 150 140 6. 0 130 120 5. 5 110 100 5. 0 90 114 4. 5 80 70 4. 0 60 3. 5 50 40 10.81030 6. 5 35.81670 N 3. 0 30 2.97 2.97 3.00 1.99 H3C 49.28530 49.14360 49.00160 48.85980 48.71710 43.26070 2.00 2.97 2.01 H3C 73.14920 13 121.73290 7. 0 130.31620 128.57620 126.27460 7. 5 135.69370 153.01930 164.75230 2.3651 3.0668 3.0199 3.3124 3.3100 4.1161 4.8117 7.5242 7.5114 7.4980 7.3874 7.3823 7.3792 7.3738 1 H NMR SPECTRUM OF NORAMINOPHENAZONE noramino-phenazon meod - 600 SO 3Na N CH3 N O 2. 5 20 ppm C NMR SPECTRUM OF NORAMINOPHENAZONE noramino-phenazon meod 600 ppm 160 150 140 130 120 110 6. 5 6.0 100 115 5.5 90 80 5. 0 70 60 36.37740 N O CH3 CH3 50 4.9904 6.9460 6.9430 6.9323 6.9292 6.7509 6.7371 7.3273 7.3177 7.2433 7.2401 7.6365 7.9007 7.8899 8.2620 8.2512 3.8448 3.7989 O 3.01 3.06 O 4.1128 4.0448 H3C 3.03 3.04 1.99 O 56.77260 56.44910 56.16130 55.73500 77.21150 76.99940 76.78810 13 105.92520 104.84030 7. 0 1.01 1.01 1.16 1.01 1.00 1.01 H3C 112.37760 111.19730 7.5 122.38170 121.44990 120.69330 129.14420 127.91900 8.0 136.80130 149.34970 148.21720 153.93940 152.45180 156.86450 1.00 1 H NMR SPECTRUM OF PAPAVERINE papaverin cdcl3 - 600 4. 5 4. 0 40 ppm C NMR SPECTRUM OF PAPAVERINE papaverin cdcl3 600 30 ppm 1 H NMR SPECTRUM OF PARACETAMOL NH 1.9911 2.5005 3.5654 6.7074 6.6940 7.3647 7.3512 9.1579 9.6582 paracetamol dmso - 600 CH3 O 9. 5 9. 0 8.5 8.0 7. 5 13 7. 0 6. 5 6. 0 5.5 5. 0 4. 5 4. 0 3.05 0.46 2.06 2.04 1.02 1.00 HO 3. 5 3.0 2. 5 2. 0 ppm C NMR SPECTRUM OF PARACETAMOL 160 150 140 130 120 110 100 90 116 80 70 60 50 40 23.75170 39.73080 39.59000 39.45080 39.31230 115.07970 121.00010 131.04160 153.22760 167.70020 paracetamol dmso 600 30 ppm 160 150 140 130 120 110 100 90 117 80 70 3. 5 60 3. 0 50 2. 5 40 30 19.21570 4. 0 25.11760 4. 5 28.24710 5. 0 38.33740 5. 5 57.59350 57.51060 55.35100 49.28150 49.14060 48.99940 48.85650 48.71490 45.14760 6. 0 61.28300 13 1.02 2.03 0.96 1.03 0.95 2.18 2.02 3.04 1.02 0.98 1.10 0.95 0.93 1.01 1.01 1.00 0.97 HO 67.90120 6. 5 102.07470 117.09970 7. 0 120.47900 123.85860 7. 5 127.36100 8. 0 131.48420 139.24550 8. 5 148.04980 147.96780 147.10910 144.56570 160.15030 1.00 8.680 7.910 7.895 7.795 7.787 7.487 7.483 7.379 7.375 7.364 7.360 6.302 5.764 5.753 5.747 5.735 5.724 5.718 5.707 5.107 5.079 5.001 4.983 4.895 4.309 4.025 3.631 3.614 3.610 3.603 3.592 3.313 3.310 3.307 3.305 3.300 3.288 3.283 3.279 3.270 3.266 3.261 3.257 2.819 2.246 2.234 2.224 2.211 2.203 2.197 2.191 2.083 2.078 1.966 1.511 1.506 1 H NMR SPECTRUM OF QUININE kinin meod - 600 H2C N H3C O 2. 0 1. 5 20 ppm kinin meod 600 C NMR SPECTRUM OF QUININE ppm 1 H NMR SPECTRUM OF SULFADIMIDINE 11. 0 10. 5 10. 0 9. 5 9. 0 8. 5 13 8. 0 7. 5 7. 0 6. 5 2.2140 6.03 3.4610 5.9610 1.98 6.6517 6.6043 6.5899 1.01 2.02 2.00 0.90 7.6939 7.6795 11.0647 sulfadimidin dmso - 600 6. 0 5. 5 5. 0 4. 5 4. 0 3. 5 3. 0 2. 5 ppm C NMR SPECTRUM OF SULFADIMIDINE O O 23.07260 39.86910 39.72970 39.59080 39.45180 39.31220 39.17300 CH3 113.74380 112.40110 111.92250 111.40600 125.10320 130.37300 152.90540 156.65140 167.31950 sulfadimidin dmso - 600 N S NH N CH3 H2N 160 150 140 130 120 110 100 90 118 80 70 60 50 40 30 ppm 119